GJRMI - Volume 2, issue 10, October 2013 by Dr. Hari Venkatesh.K.Rajaraman

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INDEX – GJRMI, Vol. 2, Iss. 10, October 2013 MEDICINAL PLANTS RESEARCH Natural Resources PHYTO-CHEMISTRY, ANTIBACTERIAL ACTIVITY AND CHROMOSOME NUMBER OF CENTAUREA SOLSTITIALIS L. GROWN IN ALGERIA Takia Lograda, Messaoud Ramdani, Pierre Chalard, Gilles Figueredo, Khadra Khalfoune, Hafsa Silini 675–684

Botany & Micro-Biology HERBAL REMEDIES FOR LEUCORRHOEA: A STUDY FROM THE GARHWAL HIMALAYA, INDIA Rana C S, Ballabha Radha, Sharma A, Dangwal L R, Tiwari J K

685–691

Botany ETHNO-BOTANICAL STUDY OF PLANTS USED FOR TREATING MALARIA IN A FOREST: SAVANNA MARGIN AREA, EAST REGION, CAMEROON BETTI Jean Lagarde, CASPA Roseline, AMBARA Joseph, KOUROGUE Rosine Liliane

692–708

INDIGENOUS MEDICINE Ayurveda – Panchakarma STANDARDIZATION OF ERANDAMOOLADI KWATHA CHURNA – FORMULATION USED IN MEDICATED ENEMA THERAPY (BASTI KARMA)

Lohith B A, Sunil Kumar K N, Girish K J

COMPOUND 709–715

Ayurveda – Stree Roga & Prasooti Tantra ROLE OF PHALAGHRITA AND UTTARBASTI IN THE MANAGEMENT OF VANDHYATVA (INFERTILITY) WITH REFERENCE TO CERVICAL FACTORS Pandya Neha R, Donga Shilpa B, Mistry I U

COVER PAGE PHOTOGRAPHY: DR. HARI VENKATESH K R, PLANT ID – INFLORESCENCE OF ERANDA (RICINUS COMMUNIS L.), OF THE FAMILY EUPHORBIACEAE PLACE – KOPPA, CHIKKAMAGALUR DISTRICT, KARNATAKA, INDIA

716–723

Research article PHYTO-CHEMISTRY, ANTIBACTERIAL ACTIVITY AND CHROMOSOME NUMBER OF CENTAUREA SOLSTITIALIS L. GROWN IN ALGERIA Takia Lograda1*, Messaoud Ramdani2, Pierre Chalard3, Gilles Figueredo4, Khadra Khalfoune5, Hafsa Silini6 1, 2, 5

Laboratory of Natural Resource Valorisation, Faculty of Natural Sciences and Life, Ferhat Abbas University, 19000 Setif, Algeria 3 Clermont Université, ENSCCF, Institut de Chimie de Clermont-Ferrand, BP 10448, F-63000 CLERMONTFERRAND, France / CNRS, UMR 6296, ICCF, F-63171 AUBIERE, FRANCE 4 LEXVA Analytique, 460 rue du Montant, 63110 Beaumont, France 6 Laboratory of Applied Microbiology, Sciences Faculty, Ferhat Abbas University, 19000 Setif, Algeria. *Corresponding author: Email: tlograda63@yahoo.fr; Phone: (213)36835894; Fax: (213)36937943.

Received: 19/08/2013; Revised: 27/08/2013; Accepted: 30/09/2013

ABSTRACT The hydrodistilation of Centaurea solstitialis, gave a yield of 0.35%. The chemical analysis of the essential oil by GC/MS, has allowed identifying 41 compounds corresponding to 99.37% of the total oil. The major compounds of the oil are n-heneicosane (17.30%), hexadecanoic acid (12.79%), n-tricosane (10.51%), n-pentacosane (5.64%) and caryophyllene oxide (5.03%). The antibacterial activity of the oil was tested on nine bacterial strains. The essential oil of this species has a moderate to significant antibacterial activity. The caryological study of C. solstitialis has allowed us to identify a karyotype with 2n = 2x = 18 chromosomes. The basic number x = 9 is reported for the first time in Algeria and Western Mediterranean clade. KEYWORDS: Centaurea solstitialis, Essential oil, Antibacterial activity, Karyology, Chromosome, Algeria

Cite this article: Takia. L., Messaoud. R., Pierre. C., Gilles. F., Khadra. K., Hafsa. S., (2013), PHYTO-CHEMISTRY, ANTIBACTERIAL ACTIVITY AND CHROMOSOME NUMBER OF CENTAUREA SOLSTITIALIS L. GROWN IN ALGERIA, Global J Res. Med. Plants & Indigen. Med., Volume 2(10): 675–684

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 10 | October 2013 | 675–684

INTRODUCTION Centaurea solstitialis L., commonly known as yellow star thistle, belongs to clade West Mediterranean (Garcia et al., 2006). It is an annual or biennial species (Quézel et Santa, 1963). C. solstitialis prefers arid lands, fields, pastures, railways, roadsides and disturbed areas (Roché and Thill, 2001). It is usually associated with human disturbance (Thomsen et al., 1996). It is an invasive plant listed as a noxious weed in the western areas of North America and is the target of classical biological control (Maddox, 1981; Rosenthal et al., 1994, Beck et al., 2008). Some species of Centaurea are used as ornamental plants. A wide range of therapeutic effects have been attributed to this genus in traditional medicines, endocrine diseases, inflammatory disorders, gastrointestinal symptoms, urogenital, cardiovascular problems, parasitic infections and microbial, anti-ulcerogenic, antioxidant, antiviral, anticancer and cytotoxic properties (Ugur et al., 2009; Ayad et al., 2012). Secondary metabolites of Centaurea, can take part in defence against herbivores or have antimicrobial activity (Ugur et al., 2009; Esmaeili and Khodadadi, 2012). These metabolites are lipophilic compounds, sesquiterpene lactones (Tarasov et al., 1975; Koukoulitsa et al., 2005); Flavonoids, essential oils and phenols (Zapesochnaya et al., 1978.). Other compounds were also isolated from Centaurea (Flamini et al., 2002; Huseyin et al., 2003; Asadipour et al., 2005; Yayli et al., 2005; Rosselli et al., 2009; Formisano et al., 2010). C. solstitialis is a well known species because of its wide geographic distribution and its medicinal properties against digestive disorders and malaria (Fujita et al., 1995; Honda et al., 1996; Yesilada et al., 2004). The aerial parts have an antinociceptive and antipyretic activities supporting affirmation of traditional folk medicine Turkish (KupeliAkkol et al., 2009).

The extract of the aerial parts of C. solstitialis showed anti-ulcer effect, a cytotoxic and antiviral activity (Yesilada et al., 2004; Özçelik et al., 2009). Ingestion of C. solstitialis by horses induced neuro-degeneration (Chang et al., 2012). Studies have revealed the presence of nitrogen compounds imparting neurotoxic effects to the plant (Moret et al., 2005). Several studies have focused on the biological effects of C. solstitialis against herbivorous insects (Fornasari et al., 1991; Maddox et al., 1996), and against fungi (Bruckart, 1989). The main active ingredients of the essential oil of C. solstitialis are hexadecanoic acid, caryophyllene oxide, bicyclogermacrene, β-eudesmol and spathulenol (Esmaeili et al., 2006; Kilic, 2013). Three sesquiterpene lactones (chlorohyssopifolin-A, chlorojanerin and 13acetyl solstitialin-A) were isolated from the aerial parts of C. solstitialis in Turkey (Özçelik et al., 2009). Earlier studies showed better antimicrobial activity against the gram-positive bacteria when compared to the gram-negative bacteria (Yayli et al., 2005). The essential oils of C. sessilis and C. armena showed antibacterial activity against Yersinia pseudotuberculosis, Enterococcus faecalis, Staphylococcus aureus, and Bacillus subtilis. However, no antimicrobial activity was observed against the other five microorganisms tested (Yayli et al., 2005). The basic chromosome numbers of the genus range from x = 7 to x = 12 (Garcia-Jacas et al., 1997; Meric et al., 2010) and it has three ploidy levels (2x, 4x and 6x) (Romaschenko et al., 2004; Siljak-Yakovlev et al., 2005). The chromosome numbers in the genus are known only for half of the species (Garcia-Jacas et al., 1997; 1998a, b; Romaschenko et al., 2004). However, the chromosome number is an important karyological feature for plant taxonomy and there is close correlation between karyology and systematics in Centaurea (Romaschenko et al., 2004).

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 10 | October 2013 | 675–684

In the present study, the aim was to identify the chemical composition of the oils of C. solstitialis obtained from plants growing in the eastern Algeria as well as to evaluate their antimicrobial activity and the identification of the chromosome number. MATERIALS & METHODS Plant material Centaurea solstitialis L. was collected from Setif locality in eastern Algeria. Aerial parts were collected during the flowering stage in June 2012. The seeds were harvested in July 2012 and were germinated in February 2013. The air dried materials were subjected to hydro-distillation for 3h using a Clevenger type apparatus. Identified by Dr. Lograda Takia, voucher specimens were deposited in the herbarium of the Department of Ecology and Biology, Setif University, Algeria. The oil obtained was collected and dried over anhydrous sodium sulphate and stored in screw capped glass vials in a refrigerator at 4–5°C prior to analysis. Yield based on dried weight of the samples was calculated. Essential oil analysis The essential oils were analysed on a Hewlett-Packard gas chromatograph Model 5890, coupled to a Hewlett-Packard model 5971, equipped with a DB5 MS column (30 m × 0.25 mm; 0.25 μm), programming from 50°C (5 min) to 300°C at 5°C/min, with a 5 min hold. Helium was used as the carrier gas (1.0 mL/min); injection in split mode (1:30); injector and detector temperatures, 250 and 280°C, respectively. The mass spectrometer worked in EI mode at 70 eV; electron multiplier, 2500 V; ion source temperature, 180°C; MS data were acquired in the scan mode in the m/z range 33–450. The identification of the components was based on comparison of their mass spectra with those of NIST mass spectral library (Masada, 1996; NIST, 2002) and those described by Adams, as well as on comparison of their retention indices either with those of authentic compounds or with literature values (Adams, 2001).

Antibacterial Activity The antimicrobial activities of the essential oils were evaluated against both Gram positive (Enterobacter cloacae ATCC 13047, MRSA (Methicillin-resistant Staphylococcus aureus), Staphylococcus aureus ATCC 25923) and six Gram negative bacteria (Escherichia coli ATCC 25922, Pseudomonas syringae, Salmonella sp, Serratia liquefaciens ATCC 27592, Serratia marcescens ATCC 14756, Shigella sp). The bacterial inoculums was prepared from overnight broth culture in physiological saline (0.8 % of NaCl) in order to obtain an optical density ranging from 0.08–01 at 625 nm. Muller-Hinton agar (MH agar) and MH agar supplemented with 5 % sheep blood for fastidious bacteria were poured in Petri dishes, solidified and surface dried before inoculation. Sterile discs (6 mm Φ) were placed on inoculated agars, by test bacteria, filled with 10 μl of mother solution and diluted essential oil (1:1, 1:2, 1:4, and 1:8 v:v of DMSO). DMSO was used as negative control. Bacterial growth inhibition was determined as the diameter of the inhibition zones around the discs. All tests were performed in triplicate. Then, Petri dishes were incubated at 37°C during 18 to 24 h aerobically (Bacteria). After incubation, inhibition zone diameters were measured and documented. Karyology For karyotypic analysis, the squashing method is used. The root-tip meristems from germinating seeds were usually used for chromosome preparations; only the root-tips of C. fontanesii were taken from wild plants in their natural habitat. A pre-treatment at room temperature for 1.5 h was usually applied before fixation of the root-tips, in a 0.05% water solution of colchicine. After fixation in a cold mixture of ethanol acetic acid (3:1), the root-tips were stored in 70° ethanol and at a low temperature, until used. The following procedure involved the maceration in 45% acetic acid for 15 min. The following procedure involved the maceration in 45% acetic acid for 15 min. Staining of chromosomes was made of emerging root-tips in acetic orcein with heating

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for one minute. Cutting off the meristems and squashing them in a drop of orcein. RESULTS The hydro-distillation of the essential oil of Centaurea solstitialis gave a viscous liquid with a color green blue. The yield of essential oil of the samples is 0.35%. The analysis and identification of the components of the essential oil was performed using the (GC-MS). The compounds identified in these oils and their relative abundances are listed in order of their appearance (table 1). The investigation allowed to identify 41 chemical compounds in the essential oil, corresponding to 99.37% of the total oil. The chemical composition of the essential oil of C. solstitialis is dominated by the presence of a major product, n-heneicosane with (17.30%), hexadecanoic acid (12.79%), ntricosane (10.51%), n-pentacosane (5.64%) and caryophyllene oxide (5.03%). Ten chemical classes have been identified in the essential oil of C. solstitialis (table 2). Alkanes, with six components, represent (36.17%), the major constituent is nheneicosane (17.30%), followed by four fatty acids (20.03%) and seven sesquiterpenes (11.82%) in which caryophyllene oxide (5.03%) was present in major amount. Other classes were also present such as monoterpenes

(6.3%), one pheromone (Z, Z) -6,9-cis-3,4epoxy-nonadecadiene with (3.58%), followed by a sesquiterpene alcohol, eudesmol (2.51%). The antimicrobial activity for the essential oils of C. solstitialis was tested in vitro using the agar-well diffusion method with the microorganisms as seen in Table 3. The essential oils showed moderate antibacterial activity against all bacteria tested. The undiluted oil showed a very significant activity against Enterobacter cloacae with an inhibition diameter of 8 mm while gentamicin had no effect. A significant antibacterial activity against MRSA, Serratia liquifaciens, Serratia marcescens and Pseudomonas aeruginosa has noticed. For dilution 1/2 the activity is zero for the other dilutions, except for Pseudomonas aeruginosa, the dilution 1/8 gives an average activity. The observation of metaphase plates of Centaurea solstitialis, showed a diploid chromosome number 2n = 2x = 18 and; with a basic chromosome number x = 9 (Figure 1). On reviewing, it was understood that no cytogenetic studies have been conducted on this species in Algeria. Chromosomes were mostly small (6 microns) and the similarity in size and shape made it difficult to distinguish between different chromosomes.

Figure 1: Metaphase plate of root meristem cells of Centaurea solstitialis 2n = 2x = 18

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Table 1: Chemical composition of Centaurea solstitialis essential oil

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41

Compounds Sabinene Limonene γ-terpinene Terpinene 4-ol 2-Cyclohexen-1-one, 2-methyl-5-(1-methylethenyl)1,1-Bicyclohexyl α-copaene β-elemene β-caryophyllene β-bergamotene trans Germacrene-D α-tridecene Δ-cadinene Elemole Lauric acid Caryophyllene oxide Dill apiole Epi-cubenol-1 α-épi muurolol Eudesmol Valencene DB5-1624 β-sesquifenchene Myrestic acid Octane, 2-methylMyristylaldehyde 2-pentadecanone-6,10,14-trimethyl 1,2-Benzenedicarboxylic acid, bis(2-methylpropyl) ester 5-Methylenebicyclo[2.2.1]hept-2-en-7-ylidene)acetic acid 2,6,10,14-Hexadecatetraen-1-ol, 3,7,11,15-tetramethyl-Ac Hexadecanoic acid MERCK 5-β, 8- β, H,9- β, H,10-α-Labd-14-ene, 8,13-epoxy 2,6,10-Dodecatrien-1-ol, 3,7,11-trimethylDehydroabietane n-heneicosane 6,9-cis-3,4-epoxy-nonadecadiene (Z,Z) 2-Methyl-Z,Z-3,13-octadecadienol Docosane n-tricosane Dotriacontane n-pentacosane 9-(2',2'-Dimethyl propanoilhydrazono)3,6-dichloro-2,7bis

KI 1029 1031 1074 1177 1290 1301 1376 1370 1421 1480 1499 1510 1524 1550 1568 1581 1622 1627 1641 1652 1663 1680 1720 1800 1880 1902 1960 1967 1972 1984 1987 1989 1990 2100 2150 1955 200 2300 3200 2500 2540

Total (%)

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TR % 6.667 3.26 7.939 0.79 8.575 0.56 11.079 0,33 12.268 0.16 12.583 0.08 14.580 036 14.794 0.21 15.312 0.43 15.809 0.02 16.289 1.65 16.436 0.54 16.869 1.78 17.328 2.70 17.537 3.08 17.846 5.03 18.334 1.63 18.512 1.37 18.738 0.95 18.915 2.51 19.138 1.28 19.700 0.93 20.341 3.28 20.838 0.60 21.069 0.72 21.379 4.10 21.605 3.77 21.916 0.39 22.249 0.68 22.973 12.79 23.357 1.16 23.639 1.03 24.067 0.66 24.564 17.30 24.992 3,58 25.476 0.71 25.687 1.39 26.773 10,51 27.824 0.73 28.817 5.64 29.084 0.65 99.37

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Table 2: Chemical classes and Compounds majority in Centaurea solstitialis Chemical classes

Number

Compounds majority

Alkanes Fatty acids Sesquiterpenes Monoterpenes Sesquiterpene alcohol Terpenes Aldehydes Alcohol

36.2 20.0 11.8 6.3 4.8 2.4 0.7 0.7

6 4 7 6 3 3 1 1

17.3 12.8 5.0 3.3 2.5 0.8 0.7 0.7

Others

13.8

n-heneicosane Hexadecanoique acid Caryophyllene oxid Sabinene Eudesmol Limonene Myristylaldehid 2-Methyl-Z,Z-3,13octadecadienol 2-pentadecanone-6, 10, 14trimethyl

4.1

Table 3: Antibacterial activity of Centaurea solstitialis essential oil Bacteria Enterobacter cloacae Salmonella sp Echerichia coli ATCC Staphylococcus aureus ATCC MRSA Serratia marcescens Serratia liquifaciens Pseudomona aeruginosa ATCC Shigella sp

Gent

0 16 14 25 14 12 10 10 15

8 7 7 8 11 12 12 10 10

1/2 0 0 0 7 7 7 9 8 8

Dilution 1/4 0 0 0 0 0 0 0 0 8

1/8 0 0 0 0 0 0 0 7 7

Gent.= Gentamicine ; MRSA = Methicillin-resistant Staphylococcus aureus; EO= Essential oil

DISCUSSION The yield of essential oil of Centaurea solstitialis was 0.35%. This yield is low compared to other herbs, such as Rosmarinus (1–2.5%) and Thymus (2–2.75%) (Edward et al., 1987). The chemical composition of C. solstitialis from Algeria differs from the region of Turkey (Kilic, 2013), with the presence of βeudesmol, bicyclogermacrene the spathulenol and germacrene-D. The oil analysis of C. solstitialis of Iran gave eighteen compounds, with the major compounds, palmitic acid and caryophyllene oxide (Esmaeili and Khodadadi, 2012). Three sesquiterpene lactones isolated from the aerial parts of C. solstitialis in Turkey

(Özçelik et al., 2009), were totally absent from the samples. The results of the essential oil are closer to the chemical composition of samples of Iran with the presence of hexadecanoic acid and caryophyllene oxide. Esmaeili and Khodadadi (2012) have found eight monoterpenes, nine sesquiterpenes and one fatty acid in the oil of C. solstitialis, while in the composition of the species we found six alkanes, four fatty acids and seven sesquiterpenes. The comparison of the results of C. solstitialis with those found in C. depressa, we noted the presence of four oxygenated monoterpenes, six sesquiterpenes hydrocarbons, ten oxygenated sesquiterpenes, five aliphatic hydrocarbons and one acid. These differences in results could be attributed to the nature of the chemical composition of the oil.

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Despite the wealth of our species in alkanes and fatty acids, the antimicrobial activity is moderate to low. This can be explained by the concentration of sesquiterpenes which play an essential role in bacterial inhibition (Esmaeili and Khodadadi, 2012). In addition the rate of aldehydes is not important enough to justify a higher activity. Tekeli et al., (2011), identified a strong antibacterial activity of C. solstitialis on Escherichia coli and Staphylococcus aureus and concluded that the essential oil of C. solstialis subsp. solstialis can be used as an antibiotic for infections due to Staphylococcus aureus.

the supplement volume flora of Turkey (Davis et al., 1988). For Centaurea tuberosa, two chromosomal number were reported, the first (2n = 22) by Siljak-Yakovlev et al., (2005) and the second by Bancheva and Greilhuber (2006) who found 2n = 20. The basic chromosome number x = 9 is not reported in the western Mediterranean, but this clade is present in the subgenus Centaurea. The number x = 9 is considered the ancestral number of the family Asteraceae (Bremer, 1994). The presence of this number x = 9 is reported in Algeria.

The observation of metaphase plates of Centaurea solstitialis, allowed us to identify a diploid chromosome number 2n = 2x = 18 and; with a basic chromosome number x = 9 (Figure 1). Cytological research on Centaurea species belonging to the flora of Turkey have shown that chromosome number is variable (2n = 16, 18, 20, 22, 24, 36, 40) (Davis et al., 1988). While those in the genre are (2n = 16, 18, 20, 30, 34, 36, 54) (Romaschenko et al., 2004).

Chemical analysis allowed us to identify the major components of Centaurea solstitialis essential oil; n-heneicosane, hexadecanoic, tricosane n-, n-pentacosane and caryophyllene oxide. The results obtained in this study are very different from the previously reported data. The antibacterial activity of our oil was tested on nine bacterial strains. The results showed that the concentrate of the essential oil of C. solstitialis has a moderate inhibitory activity against all the bacteria used. This work could provide data especially on the chromosome number of Centaurea solstitialis, we identified a diploid chromosome number (2n = 2x = 18), with a base number of x = 9, which is reported for the first time in Algeria and West Mediterranean clade.

The investigation showed that Centaurea solstitialis has a diploid chromosome number (2n = 18). This result was observed in ten meristematic cells, it is mentioned for the first time. The work on the species in the islands of Samos in Turkey reported a diploid with 2n = 16 (Georgiadis and Christodoulakis, 1984; Bancheva and Greilhuber, 2006). In Turkey Huseyin et al., (2007) reported a tetraploid with 2n = 32. The number of chromosomes in Centaurea amanicola is 2n = 16 (Davis et al., 1988). However, it was recorded as 2n = 18 in

CONCLUSION

ACKNOWLEDGEMENTS The work was supported by VRBN laboratory, Setif University, Algeria and Chemical Laboratory of carbohydrates Heterocyclic of Clermont Ferrant, France.

REFERENCES Adams RP (2001). Identification of essential oils components by Gas Chromatography-Mass spectroscopy., Allured Publishing Corporation Carol Stream, Illinois USA.

Asadipour A, Mehrabani M, Najafi M (2005). Volatile oil composition of Centaurea aucherie (DC.) Wagenitz, Daru. 13 (3): 160â&#x20AC;&#x201C;164.

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Ayad R, Ababsa Z, Belfadel F, Akkal S, León F, Brouard I, Medjiroubi K, (2012). Phytochemical and biological activity of Algeian Centaurea melitensis, Int. J . Med. Arom. Plants, 2 (1): 151–154. Bancheva S. and Greilhuber J., 2006; Genome size in Bulgarian Centaurea s.l. (Asteraceae), Pl Syst Evol., 257(1-2): 95–117. Beck John J, Lincoln Smith, Glory B Merrill (2008). In Situ Volatile Collection, Analysis, and Comparison of Three Centaurea Species and Their Relationship to Biocontrol with Herbivorous Insects, J. Agric. Food Chem., 56: 2759–2764. Bremer K (1994). Asteraceae Cladistics and classification, Ed. Timber Press Portland, USA. Bruckart W (1989). Host range determination of Puccinia jaceae from yellow starthistle. Plant Disease, 73: 155–160. Chang HT, Rumbeiha WK, Patterson JS, Puschner B, Knight AP (2012). Toxic Equine Parkinsonism: An Immunohistochemical Study of 10 Horses with Nigropallidal Encephalomalacia, Veterinary Pathology, 49(2): 398–402. Davis PH, Mill RR, Tan K (1988). Flora of Turkey and the East Aegean Islands, Vol. 10, Supplement. Edinburgh University Press, Edinburgh. Edward PC, Varro ET, Lynn RB (1987). Pharmacognosy. Ed. LEA and Febiger, 18 tome, pp 184–187. Esmaeili A, Khodadadi E (2012). Volatile compounds of essential oil Centaurea behen L. grown in Iran, Journal of Paramedical Sciences (JPS), 3(2): 8–11. Esmaeili A, Rustaiyan A, Akbari MT, Moazami N, Masoudi S, Amiri H (2006). Composition of the Essential Oils of Xanthium strumarium L. and Cetaurea solstitialis L. from Iran,

Journal of Essential Oil Research, 18(4): 427–429. Flamini G, Ertugrul K, Cioni PL, Morelli I, Dural H, Bagci Y (2002). Volatile constituents of two endemic Centaurea species from Turkey: C. pseudoscabiosa subsp. pseudoscabiosa and C. hadimensis, Biochemical Systematics and Ecology, 30: 953–959 Formisano C, Senatore F, Bancheva S, Bruno M, Rossellic S (2010). Volatile components from aerial parts of Centaurea spinosociliata Seenus ssp. cristata (Bartl.) Dostál and Centaurea spinosociliata Seenus ssp. spinosociliata growing wild in Croatia., Croatica Chemica Acta, 84(4): 403– 408. Fornasari L, Turner C, Andres L (1991). Eustenopsis villosus (Coleoptera: Curculionidae) for biological control of yellow starthistle (Centaurea solstitialis) in North America. Environmental Entomology, 20: 1187– 1194. Fujita T, Sezik E, Tabata M, Yesilada E, Honda G, Takeda Y, Takaushi Y, Tanaka T (1995). Traditional medicine in Turkey VII Folk medicine in middle and west black Sea Regions, Economic Botany, 49: 406–422. Garcia-Jacas N, Uysal T, Romaschenko K (2006). Centaurea revisited a molecular survey of the Jacea group. Annals of Botany, 98: 741–753. Garcia-Jacas N, Susanna A, Mozaffarian V (1998b). New chromosome counts in the subtribe Centaureinae (Asteraceae, Cardueae) from West Asia., III., Bot. J. Linn. Soc., 128(4): 413–422. Garcia-Jacas N, Susanna A, Ilarslan R, Ilarslan H (1997). New chromosome counts in the subtribe Centaureinae (Asteraceae, Cardueae) from West Asia. Bot. J. Linn. Soc., 125(4): 343–349.

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Garcia-Jacas N, Susanna A, Vilatersana R, Guara M (1998a). New chromosome counts in the subtribe Centaureinae (Asteraceae, Cardueae) from West Asia, II., Bot. J. Linn. Soc., 128(4): 403–412. Georgiadis T, Christodoulakis D (1984). Contribution à l’étude cytogéopraphique des Centaurées de I’île de Samos. Candollea, 39: 307– 318. Honda G, Yesilada E, Tabata M, Sezik E, Fujita E, Takeda Y, Takaushi Y, Tanaka T (1996). Traditional medicine in Turkey VI. Folk medicine in West Anatolia : Afyon, Kutahya, Denizli, Mugla, Aydin provinces, Journal of Ethno pharmacology, 53: 75–87. Huseyin Dural, Yavuz Bagci, Kuddisi Ertugrul, Hakki Demirelma, Guido Flamini, Pier Luigi Cioni, Ivano Morelli (2003). Essential oil composition of two endemic Centaurea species from Turkey, Centaurea mucronifera and Centaurea chrysantha, collected in the same habitat, Biochemical Systematics and Ecology, 31: 1417–1425 Huseyin I, Hayırlıoglu-Ayaz S, Ozcan M (2007). Chromosome numbers of the twenty-two Turkish plant species, Caryologia, 60(4): 349–357. Kilic O (2013). Essential oil compounds of three Centaurea L. taxa from Turkey and their chemotaxonomy. Journal of Medicinal Plants Research, 7(19): 1344–1350. Koukoulitsa C, Geromichalos GD, Skaltsa H (2005). Vilsurf analysis of pharmacokinetic properties for several antifungal sesquiterpene lactones isolated from Greek Centaurea sp., Journal of Computer-aided Molecular Design, 19: 617–623.

Küpeli-Akkol E, Arif R, Ergun F, Yesilada E (2009. Sesquiterpene lactones with antinociceptive and antipyretic activity from two Centaurea species, Journal of Ethno pharmacology, 122(2): 210–215. Maddox DM (1981. Introduction, Phenology and Density of Yellow Starthistle in Coastal, Intercoastal, and Central Valley Situations in California, U.S. Department of Agriculture, Agricultural Research Service, ARR-W-20, pp 1–33. Maddox DM, Joley DB, Supkoff DM, Mayfield A (1996). Pollination of yellow starthistle (Centaurea solstitialis) in California, Canadian Journal of Botany, 74: 262–267. Masada Y (1996). Analysis of essential oils by Gas Chromatography and Mass Spectrometry. J. Wiley & Son’s, Inc. New York. Meriç Ç, Arda H, Güler N, Dayan S (2010). Chromosome number and nuclear DNA content of Centaurea kilaea (Asteraceae) an endemic species from Turkey, Phytologia Balcanica, 16(1): 79–84. Moret S, Populin T, Conte LS, Cosens G (2005). HPLC determination of free nitrogenous compounds of Centaurea solstitialis (Asteraceae), the cause of equine nigropallidal encephalomalacia, Toxicon, 46(6): 651–657. NIST (2002). Mass Spectral Search Program for the NIST/EPA/NIH Mass Spectral Library, vers. 2.0. fiveash data, USA. Özçelik B, Gürbüz I, Karaoglu T, Ye_ilada E (2009). Antiviral and antimicrobial activities of three sesquiterpene lactones from Centaurea solstitialis L. ssp. solstitialis extract. Microbiol. Res., 164(5): 545–552. Quézel P, Santa S (1963). Nouvelle Flore de l’Algérie et des Régions Désertiques et Méridionales, Tome II, Ed. CNRS, Paris.

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Roché CT, Thill DC (2001). Biology of common crupina and yellow starthistle, two Mediterranean winter annual invaders in western North America, Weed Sci, 49: 439–447.

Tekeli Y, Sezgin M, Aktumsek A, Guler GO, Sandra MA (2010). Fatty cid composition of six centaurea Species growing in Konya, Turkey., Natural Product Research, 24: 1883–1889.

Romaschenko K, Ertugrul K, Susanna A, Garcia-Jacas N, Uysal T, Arslan E (2004). New chromosome counts in the Centaurea Jacea group (Asteraceae, Cardueae) and related taxa., Botanical Journal of the Linnean society, 145: 345–352.

Thomsen CD, Williams WA, Vayssieres MP, Turner CE, Lanini WT (1996). Yellow starthistle biology and control, University of California Publication, Oakland.

Rosenthal SS, Davarci T, Ercis A, Platts B, Tait S (1994). Turkish herbivores and pathogens associated with some knapweeds (Asteraceae: Centaurea and Acroptilon) that are weeds in the United States. Proc. Entomol. Soc. Wash., 96: 162–17. Rosselli S, Bruno M, Maggio A, Raccuglia R, Bancheva S, Senatore F, Formisano C (2009). Essential oils from the aerial parts of Centaurea cuneifolia Sibth. & Sm. and C. euxina Velen., two species growing wild in Bulgaria, Biochemical Systematics and Ecology, 37: 426–431. Siljak-Yakovlev S, Solic ME, Catrice O, Brown SC, Papes D (2005). Nuclear DNA content and chromosome number in some diploid and tetraploid Centaurea (Asteraceae: Cardueae) from the Dalmatia region, Pl. Biol, 7(4): 397–404. Tarasov VA, Kasymov SZ, Sidyakin GP (1975). The isolation of Cnicin from Centaurea squarrosa., Chemistry of natural Compounds, 9: 414

Source of Support: University, Algeria;

VRBN

laboratory,

Ugur A, Sarac N, Ceeylan O, Duru M (2009). Chemical composition of endemic Centaurea austro-anatolica and studies of its antimicrobial activity against multi-resistant bacteria, Acta Pharm, 59: 463–472. Yayli Nurettin, Ahmet Yasar, Canan Gulec, Asu Usta, Sevgi Kolayli, Kamil Coskuncelebi, Sengul Karaoglu (2005). Composition and antimicrobial activity of essential oils from Centaurea sessilis and Centaurea armena, Phytochemistry, 66: 1741–1745 Yesilada E, Gurbuz I, Bedir E, Tateli I, Khan IA (2004). Isolation of anti-ulcerogenic sesquiterpenes lactones from Centaurea solstitialis L. ssp. Solstitialis through bioassay-guided fractionation procedures in rats, Journal of Ethno pharmacology, 95: 213–219. Zapesochnaya GG, Evstratova RI, Mukhametzhanov MN (1978). Methoxyflavones of some species of Centaurea, Chemistry of Natural Compounds, 13: 590–591.

Setif

Conflict of Interest: None Declared

Chemical Laboratory of carbohydrates Heterocyclic of Clermont Ferrant, France

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Research article HERBAL REMEDIES FOR LEUCORRHOEA: A STUDY FROM THE GARHWAL HIMALAYA, INDIA Rana C S1, Ballabha Radha2*, Sharma A3, Dangwal L R4, Tiwari J K5 1, 2, 3, 4, 5

Department of Botany and Microbiology, HNB Garhwal University, Srinagar Garhwal - 246 174, Uttarakhand, India * Corresponding author: E-mail: radhekuniyal.2007@rediffmail.com

Received: 03/09/2013; Revised: 16/09/2013; Accepted: 20/09/2013

ABSTRACT In the study, an attempt has been made to document the indigenous uses and practices of plant species utilized as herbal remedies for the treatment of leucorrhoea by the inhabitants of Garhwal Himalaya. The present study provides 24 significant medicinal plants belonging to 23 genera and 17 families used as herbal remedies for the treatment of leucorrhoea. It is observed that mostly, the underground parts (roots, tubers, bulbs, rhizomes, etc.) are being used in the preparation of remedies taken either singly or mixed along with water. A list of plant species along with their family, vernacular name, life form, specimen voucher number, plant part(s) used, method of preparation and dosage pattern of the herbal remedies are provided. KEYWORDS: leucorrhoea, herbal remedies, rural women, Garhwal Himalaya.

Cite this article: Rana. C. S., Ballabha Radha., Sharma. A., Dangwal. L. R., Tiwari. J. K., (2013), HERBAL REMEDIES FOR LEUCORRHOEA: A STUDY FROM THE GARHWAL HIMALAYA, INDIA, Global J Res. Med. Plants & Indigen. Med., Volume 2(10): 685â&#x20AC;&#x201C;691

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INTRODUCTION The plants are still serving as remedies for various ailments in crude form, as modern medicine has not adequately armed the therapeutic arsenal of the natives of remote areas (Tiwari et al., 2010a). Approximately 80% of world population in developing countries depends on traditional medicines for primary healthcare (WHO, 2002) and in modern medicine too, nearly 25% are based on plant-derived drugs (Tripathi, 2002). In India, people living in villages and far flung areas depend largely on forest resources for maintaining their day-to-day needs like medicine, food, fuel and household articles (Chhetri et al., 2005), as plants have been playing a vital role in the socio-economic development of such regions (Ballabha et al., 2013a). The Indian Himalaya occupies a special place in the mountain ecosystems of the world. These geo-dynamically young mountains are not only providing life by giving water to a large part of the Indian subcontinent, but also supporting a rich variety of flora, fauna, human communities and cultural diversity (Gaur, 1999). Garhwal Himalaya occupies an important place in Indian subcontinent due to its peculiar topography, vegetation, people and traditions. About 80% of the total population is rural. The inhabitants have their own cultures, medicines, foods, etc. and are well versed with valuable knowledge accumulated through a long period of experience. Even now the inhabitants of the region are dependent on the natural resources from the forests and alpine meadows (pastures) for their sustenance and for the treatment of various ailments (Rana et al., 2010). In the remote areas traditional customs and beliefs are still maintained and modem trends are yet to reach, which provide interesting scope of ethno-botanical studies (Tiwari et al., 2010a). Many works have been done on folk medicines and ethno-medicinal plants used by the inhabitants of Garhwal Himalayan region for various ailments (Singh and Bisht, 1993;

Samant et al., 1996; Maikhuri et al., 1998; Tiwari et al., 2010bc; Rana et al., 2012, 2013; Ballabha et al., 2013b). However, no attention has been paid on documentation of plants used in the treatment of leucorrhoea. Leucorrhoea is quite common in the women of Garhwal Himalaya as well as all over the globe. Leucorrhoea, commonly known as Swet Pradar and Safed Pani, refers to a whitish discharge from the female genital organs. It is an abnormal condition of the reproductive organs of the women. Consequently, the local inhabitants used various types of plants available in their surrounding for the treatment of leucorrhoea on intimation of faith herbal healers. Documentation of such practices is required in view of gradual disappearance of this knowledge in new generations (Tiwari et al., 2010a). Therefore, an attempt has been made to document herbal remedies for leucorrhoea from the remote areas of Garhwal Himalaya, India. MATERIALS AND METHODS Extensive field surveys were made in the remote areas of Garhwal Himalaya during the last 10 years for the survey of the vegetation and ethno-medicinal uses. A structured questionnaire was used to collect data on local name of plants, uses, parts used and mode of application. Ethno-medicinal information on plants was collected through interviewing local communities particularly from faith herbal healers (Vaidhyas), women, peasants, shepherds and priests. To determine the authenticity of information collected during field work, repeated verification of data from different informants was done. Thus, only the specific and reliable information, cross-checked with informants has been incorporated in the present study. The recorded plant species were identified with the help of Garhwal University Herbarium (GUH), Herbarium of the Botanical Survey of India Northern Circle Dehradun (BSD) and regional Floras (Duthie, 1906; Osmaston, 1927; Rau, 1961; Naithani, 1984-85; Hajra and Balodi, 1995; Gaur, 1999). The plant species have been deposited in the Herbarium

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of Hemwati Nanadan Bahuguna Garhwal University (GUH), Srinagar Garhwal, Uttarakhand (India). RESULTS AND DISCUSSION In the present study, a total number of 24 valuable medicinal plants have been documented for the treatment of leucorrhoea. The recorded plant species belong to 23 genera and 17 families. The plant species along with their family, vernacular name, life form,

specimen voucher number, plant part(s) used, method of preparation and dosage pattern of the herbal remedies have been presented in Table 1. Some important medicinal plants used by local inhabitants to cure leucorrhoea have been given in Figure 1. Herbs were the primary source of medicines in terms of the number of species (20) followed by trees (02), shrubs and climbers (one species each). Among the documented species, 07, 02, 15 were having alpine, sub-alpine and temperate habitat, respectively.

Table 1. List of medicinal plants used for the treatment of leucorrhoea in Garhwal Himalaya. Botanical Name

Family/Habit/ Habitat

Vernacular Name

GUH No.

Parts used, method of preparation and dosage pattern Roots are shade dried and powdered. The powder is taken half teaspoonful twice a day early in the morning and at night after meals up to three months.

Aconitum heterophyllum Wall. ex Royle

Ranunculaceae/H/ Alpine

Atish

19377

Aesculus indica (Wall. ex Camb.) Hook. f. Ajuga parviflora Benth.

Hippocastanaceae/ T/ Temperate Lamiaceae/H/ Temperate

Panger

19338

Seeds powder is taken ½ teaspoonful twice a day for a month.

Neelkanthi

19425

Leaf powdered is taken a ¼ teaspoonful in empty stomach twice a day early in the morning and after meals in the night for a month.

Arnebia benthamii (Wall. ex G. Don) L.M. Johnston Asparagus filicinus Buch.-Ham. ex D. Don

Boraginaceae/H/ Alpine

Balchhari

19270

Liliaceae/ S/ Temperate

Jhiran

19262

Astragalus condoleanus Royle ex Benth.

Fabaceae/H/ Alpine

Rudravanti

19436

Bombax ceiba L.

Bombacaceae/T/ Temperate

Simul

19518

Centella asiatica (L.) Urban

Apiaceae/H/ Temperate

Pan Brahmi

19583

Coleus forskohlii (Willd.) Briq.

Lamiaceae/H/ Temperate

Fiwain

19211

Corydalis meifolia Wall.

Fumariaceae/H/ Alpine

6199

Rhizomatous root powdered is taken ½ teaspoonful twice a day for a month. Extract of rhizomatous root is taken half teaspoonful in empty stomach early in the morning and after meals at night twice a day for a month. Roots are powdered and made into pills. One pill is taken twice a day, for 30 – 60 days. Approximately two grams of powdered root is taken three times a day up to 45–90 days. Aqueous extract (½ teaspoonfuls) of herbs is taken twice a day, early in morning and at night after meals for 14–28 days. Decoction (½ teaspoonfuls) of roots along with honey is taken twice a day for 3–4 weeks. Root extract is taken twice a day for a long time.

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Dactylorhiza hatagirea (D. Don) Soo

Orchidaceae/H/ Alpine

Hattajadi

19271

Heracleum lanatum Michaux, Fl. Box.

Apiaceae/H/ Temperate

Kshtra

19356

Paeonia emodi Wall. ex Royle

Paeonaceae/H/Sub Alpine

Chandra

6143

Podophyllum hexandrum Royle

Podophyllaceae/H/S ub Alpine

Shon-kakadi

19282

Rhizome powder (2.5 gram) is taken twice a day, early in the morning before meals and at night after meals for 14–28 days.

Polygonatum cirrhifolium (Wall.) Royle

Liliaceae/H/ Temperate

Teetu

19382

Root sap (5–10 ml.) along with root sap of Polygonatum verticillatum is taken twice a day for 30–45 days.

Polygonum polystachyum Wall. ex Meissn.

Polygonaceae/H/ Alpine

Kukhunya jhad

19155

Rheum moocroftianum Royle

Polygonaceae/H/ Alpine

Archu

19188

Rumex nepalensis Sprengel

Polygonaceae/H/ Temperate

Kukhunya

19250

Satyrium nepalense D. Don Saussurea gossypiphora D. Don

Orchidaceae/H/ Temperate Asteraceae/H/ Alpine

Musali

19208

Phen Kaun

19542

Root extract is taken half teaspoonful twice a day, in the morning and at night after meals for 14–28 days. Root powder (approximately half teaspoonful) is taken twice a day for one month. Extract of root is taken half teaspoonful twice a day, in the morning and at night after meals for 7–21 days. Roots juice (5–10 ml.) is taken twice a day, for a month. Roots and flowers powder (½ teaspoonfuls) is taken twice a day, early in morning and at night after meals for 15–45 days.

Smilax aspera L.

Smilacaceae/Cl/ Temperate Orchidaceae/H/ Temperate

Kukur dad

19312

Garud Panja

19280

Swertia paniculata Wall.

Gentianaceae/H/ Temperate

Chiratu

19149

Viola canescens Wall.

Violaceae/H/ Temperate

Amoya

19284

Spiranthes sinensis (Per.) Ames

Extract of the roots is taken twice a day early in the morning and at the night after meals for 2–3 months. Root dried in shade, powdered and mixed with honey, made into paste. The paste is taken approximately ½ teaspoonfuls twice a day, for a month. Leaves are dried in shade, washed with hot water thrice, and then used as vegetable twice a day for 30–60 days.

Root extract is given twice a day for a long time. Root extracted with tubers of Gymnadenia orchidis is given (half teaspoonful) twice a day in early morning and at night after meals for 4–6 weeks. The powder along with other Swertia spp. is taken ½ teaspoonful twice a day for 5–7 weeks. Extract of the whole plant is taken twice (half teaspoonful) a day, early in morning and at night after meals for 14–28 days.

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Figure 1: Some important medicinal plants used by local inhabitants to cure leucorrhoea.

A. Aconitum heterophyllum B. Aesculus indica C. Arnebia benthamii D. Saussurea gossipiphora E. Rheum moocroftianum F. Podophyllum hexandrum G. Paeonia emodi H. Polygonatum cirrhifolium I. Dactylorhiza hattagirea J. Spiranthes sinensis It was observed that, mostly the underground parts (roots, tubers, bulbs, rhizomes, etc.) of the species were used in the preparation of remedies taken either singly or mixed along with water. The patient is observed deeply before the treatment and then prescribed the medicines to treat the disease by the faith herbal healers (Rana et al., 2013). Generally, medicines were prepared in the form of powder, decoction, infusion, paste, pill, etc. Medicine is taken in the form of liquid (5 ml), powder (0.5 g) and locally made pills. Prepared medicines are usually taken along with water twice or thrice a day after or before the meals. The duration of treatment mostly depends on the effectiveness of the drugs, or it depends on

the condition of patients, which varied from weeks (4–6) to months (3–6). CONCLUSION Thus, the present study provides comprehensive information on the ethno– medicinal plants and their indigenous uses to cure leucorrhoea from the remote areas of Garhwal Himalaya, India. Based on the results, it can be concluded that the area has high potential of ethno–medicinal plant species. Moreover, the subject matter is too hard to collect the information from the patients of leucorrhoea. Therefore, there is an urgent need to educate and bring awareness in the local

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communities through meetings, awareness and training programs at village or regional level. It is also suggested that these remedies should be verified and authenticated scientifically through biological and pharmaceutical screening in different National and International laboratories.

ACKNOWLEDGEMENTS The authors are grateful to the faith herbal healers and local inhabitants of Garhwal Himalayan region for providing valuable information. C S Rana is also thankful to Prof. C M Sharma, Department of Botany, HNB Garhwal University, Srinagar Garhwal for providing Laboratory facilities.

REFERENCES Ballabha R, Singh D, Tiwari J K, Tiwari P (2013a). Diversity and Availability Status of Ethno-Medicinal Plants in the Lohba Range of Kedarnath Forest Division (KFD), Garhwal Himalaya. Global J. Res. Med. Plants & Indigen. Med., 2(4): 198–212 Ballabha R, Tiwari J K, Tiwari P (2013b). Medicinal Plant Diversity in Dhundsir Gad Watershed of Garhwal Himalaya, Uttarakhand, India. The Journal of Ethnobiology and Traditional Medicine. Photon 119, 424–433.

Maikhuri R K, Nauityal S, Rao K S, Saxena K G (1998). Role of medicinal plants in the traditional health care system: a case study from Nanda Devi Biosphere Reserve, Current Science, 75: 152–157. Naithani B D (1984-85). Flora of Chamoli. 2Vols. Botanical Survey of India, Howrah, India. Osmaston A E (1927). A Forest Flora for Kumaun. Government Press, Allahabad. Reprint 1990, Bishan Singh Mahendra Pal Singh, Dehradun, India.

Chhetri D R, Basnet D, Chiu P F, Kalikotay S, Chhetri G, Parajuli S (2005). Current status of ethnomedicinal plants in the Darjeeling Himalaya. Current Science, 89(2): 264–268.

Rana C S, Rana V, Bisht M P S (2010). An unusual composition of plant species towards zone of ablation (Tipra glacier), Garhwal Himalaya. Current Science, 99: 574–577.

Duthie J F (1906). Catalogue of plants of Kumaon and of the adjacent portions of Garhwal and Tibet based on the collections made by Strachey and Winterbottom during the years 18461849. London. Reprint 1994, Bishan Singh Mahendra Pal Singh, Dehradun, India.

Rana C S, Tiwari J K, Dangwal L R, Sundriyal R C (2012). Herbal remedies for sexual capability. Indian Journal of Traditional Knowledge. 11(4): 646– 651.

Gaur R D (1999). Flora of the District Garhwal, North West Himalaya (with Ethnobotanical Notes). Transmedia: Srinagar Garhwal, Uttarakhand, India. Hajra P K, Balodi B P (1995). Plant Wealth of Nanda Devi Biosphere Reserve, BSI, Howrah, India.

Rana C S, Tiwari J K, Dangwal L R, Gairola S (2013). Faith herbal healer knowledge document of Nanda Devi Biosphere Reserve, Uttarakhand, India. Indian Journal of Traditional Knowledge. 12(2): 208–214. Rau M A (1961). Flowering plants and ferns of north Garhwal, Uttar Pradesh, India. Bull. Bot. Surv. India, 3: 215–251.

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Samant S S, Dhar U, Rawat R S (1996). Natural resources use by some natives within Nanda Devi Biosphere Reserve in West Himalaya. Ethnobotany, 8: 40– 50. Singh H, Bisht G (1993). Traditional therapy among the Tolchhas of Chamoli district, Garhwal, UPJ, Scient Res Pl Med, 13: 7. Tiwari J K., Radha Ballabha, Tiwari P (2010a). Ethnopaediatrics in Garhwal Himalaya, Uttarakhand, India (Psychomedicine and Medicine). New York Science Journal, 3(4): 123–126. Tiwari J K, Dangwal L R, Rana C S, Tiwari P, Radha Ballabha (2010b). Indigenous uses of plant species in Nanda Devi Biosphere Reserve, Uttarakhand, India. Report and Opinion, 2(2):58–61.

Source of Support: Nil

Tiwari J K, Radha Ballabha, Tiwari P (2010c). Diversity and Present Status of Medicinal Plants in and around Srinagar Hydroelectric Power Project in Garhwal Himalaya, India: Needs for Conservation. Researcher, 2(2):50–60. Tripathi G (2002). Indigenous Knowledge and Traditional Practices of Some Himalayan Medicinal Plants. In: Samant SS, Dhar U, Palni LMS (eds) Himalayan Medicinal Plants Potential and Prospects, Gyanodaya Prakashan, Nainital, pp 151–156. WHO (2002). WHO Traditional Medicine Strategy 2002–2005. World Health Organization, Geneva.

Conflict of Interest: None Declared

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Research article ETHNO-BOTANICAL STUDY OF PLANTS USED FOR TREATING MALARIA IN A FOREST: SAVANNA MARGIN AREA, EAST REGION, CAMEROON BETTI Jean Lagarde1*, CASPA Roseline2, AMBARA Joseph3, KOUROGUE Rosine Liliane4 1

Department of Botany, Faculty of Sciences, University of Douala, BP 24 157 Cameroon IRAD, Yaoundé, Cameroon 3 Ministry of Environment, Nature protection and Sustainable development, Yaoundé, Cameroon 4 Ministry of Forestry and Wildlife, Cameroon *Corresponding Author: E-mail: lagardebetti@yahoo.fr; Phone: 00 (237) 77 30 32 72 2

Received: 26/08/2013; Revised: 30/09/2013; Accepted: 01/10/2013

ABSTRACT Ethno-botanical surveys were conducted in Andom, a village situated in a forest-savanna contact zone from December 2011 to April 2012 with the aim to gather plants that are used in traditional medicine. The method used is direct interviews conducted among adult people, mainly women. The 36 persons interviewed prescribed a total of 219 citations and 94 recipes of 59 plant species distributed in 49 genera and 27 families in the treatment of malaria or fever. About 51.6 % of the citations are made of combination of two, three; four, five, six, or seven plant species. Leaves are the plant parts that are largely used; decoctions are the pharmaceutical forms that are more cited; and recipes are essentially administered orally. A total of 29 plant species (57%) used by Andom people against malaria are also known in other regions of Cameroon and other African countries for the same use. Among these, eight plant species representing 27.6 % are well recognised in the literature for their real activity against malaria including: Alstonia boonei, Carica papaya, Citrus limon, Cymbopogon citratus, Enantia chlorantha, Morinda lucida, Picralima nitida, and Vernonia amygdalina. The fact that some plant species cited by Andom people are well recognized for their activity against Plasmodium, is a credibility index which can be attributed to the pharmacopoeia of those people on one hand and illustrates the efficiency of the method used to identify medicinal plants of the Andom village on the other hand. Future studies should be directed towards implementing strategies and programmes to identify active chemical substances of other plant species which have not yet been investigated for their chemical and anti-malarial activities in the region. KEY WORDS: Forest-savanna contact zone, Medicinal plants; Malaria; Recipe; Andom village.

Cite this article: Betti. J. L., Caspa. R., Ambara. J., Kourogue. R. L., (2013), ETHNO-BOTANICAL STUDY OF PLANTS USED FOR TREATING MALARIA IN A FOREST: SAVANNA MARGIN AREA, EAST REGION, CAMEROON, Global J Res. Med. Plants & Indigen. Med., Volume 2(10): 692–708

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INTRODUCTION Malaria is a global disease that is predominant in the tropics and caused by blood parasites, Plasmodium falciparum, Plasmodium ovale, Plasmodium malariae, and Plasmodium vivax. The parasite is transmitted to its human hosts via various mosquito species of the genus Anopheles. Malaria has a great morbidity than any other infectious diseases of the world as well as a contributing factor to poverty in tropical and subtropical regions such as subSaharan Africa (World Malarial Report, 2008). Plasmodium falciparum; the pathogenic most widespread human malaria is becoming increasingly resistant to anti-malarial drugs. The malaria parasite has gradually developed resistance to the most commonly used medicines. The resistance of Plasmodium spp. to drugs such as chloroquinone has become a serious problem in areas of endemic malaria such as Cameroon, and in malaria-free areas with occasional imported cases. This requires extra effort and continuous search for new drugs, especially with new mode of action (Muregi et al. cit. Saotoing et al., 2011; Oketch-Rabah and Mwangi, 1998). Ethnobotanical survey is an important step in the identification, selection and development of the therapeutic agents from medicinal plants (Balick, 1985, 1990, 1994; Cotton, 1996; King and Tempesta, 1994). This paper aims to analyze the traditional use of medicinal plants in the treatment of malaria in Andom, a village situated in the forest-savanah contact zone, East region, Cameroon. MATERIAL AND METHODS The study site Andom village is in the Eastern region, in the Lom and Djerem division, Diang subdivision or commune. The village was established in 1925 and is located at about 45 km from Bertoua, the regional capital of East Cameroon. Houses line both sides of National Route 1, which is 3.5 km, East to West. The population of Andom village is about 2,500. The Bamvélé people are classified within the Tuki, Bantou group, and along with the Baka and Bororo peoples, live in Andom

village. Among them, the Bamvélé people are the most prevalent ethnicity within Andom village. Andom is located at the forest-savanna transition zone, with the savanna being the main useful lands. Cassava, groundnuts, maize and cocoyams seem to be in this order, the most important crops cultivated in this savanna area. But some people are moving more and more in the forest zone in search of new and fertile soils for cultivation. The mixed cropping of cassava and groundnuts or maize under grass fallow is the most common cropping system used in Andom village. In this fallow, the wild plant species Chromolaena odorata, known locally as “Bokassa” abunds. Non-timber forest products including wild fruits (moabi, bush mango), caterpillar (egbagéndong), bushmeat (grass-cutter, rats, duikers) are used in the daily diet of the villagers as sources of complementary proteins. Andom village is rich in medicinal plants which are used for the daily healthcare. Ethno-botanical survey Data for this study were obtained from direct interviews with the local people conducted from December 2011 to April 2012 in Andom village. The survey aimed at identifying plants used in the popular pharmacopoeia among local people. The household was considered as the sample unit. In each household data were mostly recorded from adult women (mothers), because they usually knew the plants better than men and younger people. They provided useful and firsthand information on the popular use of medicinal plants. During the survey, we made enquiry “as to what ailments were treated by which plant species” rather than asking “which plants were used to treat which ailments”. For each health problem cited, the name of the plants and the plant parts used were carefully recorded. For each health problem cited, details of prescriptions (plant part used, mode of preparation, etc.) were carefully recorded. The vernacular names of the plants were recorded as much as possible, and the plants mentioned by the informants were collected. The final

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 10 | October 2013 | 692â&#x20AC;&#x201C;708

identification of plants was made at National Herbarium of Cameroon Yaounde (YA) with the help of Dr. Onana and Mr. Paul Mezili. Voucher herbal specimens, collected in three samples are kept at the YA. The therapeutic statements were made of a specific disease, a symptom or a physiological effect. Information on the diagnosis of ailments was provided through a semi-structured interview of nurses or local health officials. In this paper, anti-malarial plants refer to the plants used for treating malaria or fever on a broader scale. RESULTS List of anti-malarial plants A total of 36 persons (Table 1) provided information on the use of medicinal plants in treatment of malaria comprising 29 women and 7 men. The average age of the informant is 54 years old. A total of 51 plant species were cited for which a total of 219 citations were made on malaria (Table 2). The plant species cited are

distributed in 49 genera and 27 families. The most cited plant species are: Alstonia boonei (24 citations), Enantia chlorantha (22), Rauvolfia vomitoria (13), Dichrocephala integrifolia (12), Carica papaya (10), Citrus limon (10), Schumanniophyton magnificum (9), and Capsicum frutescens (9). The most represented families are Asteraceae (7 plant species) and Apocynaceae (5). The most cited families are Apocynaceae (52 citations), Asteraceae (31), Annonaceae (25), Rubiaceae (15), Rutaceae (11), Solanaceae and Caricaceae (10 citations each). The list of the 219 citations of anti-malarial plants recorded in Andom village is presented in table 3. Each citation or line in the table presents for a given plant species, the scientific name, the associated plant (s), the plant part cited, the mode of preparation, the voice (way) of administration, and the code of informant(s) who indicated the recipe in brackets. The first letter of the code refers to the gender (M: male, F: female), the number indicates the order number of the informant in each gender.

Table 1: List of informants Code_informant

Age

Code_informant

Age

F19

F20

F21

F22

F23

F24

F25

F26

F27

F10

F28

F11

F29

F12

F13

F14

F15

F16

F17

F18

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 10 | October 2013 | 692–708

Table 2: List of plant species cited as anti-malarials in Andom village Scientific Name Acmella caulirhiza Del. (syn. : Spilanthes filicaulis, S. africana) Ageratum conizoides L. Albizia adianthifolia (Schum.) W.F.Wight Alchornea cordifolia (Sch. & Thonn.) Müll. Arg. Alstonia boonei De Wild. Annona muricata L. Anonidium mannii (Oliv.) Engl. & Diels Beilschmiedia sp Bidens pilosa L. Bridelia scleroneura Capsicum frutescens L. Carica papaya L. Chenopodium ambrosioides L. Chromolaena odorata (L.) R. King & H. Robinson Citrus limon L. Citrus reticulata L. Clerodendrum splendens G. Don Coffea canephora Froehn. (syn : Coffea robusta Linden) Cymbopogon citratus (DC.) Stapf Dacryodes edulis (G. Don) H. J. Lam Dichrocephala integrifolia (L. f.) O. ktze Elaeis guineensis Jacq. Enantia chlorantha Oliv. Eucalyptus camaldulensis Ipomoea involucrata Beauv. Khaya ivorensis Lippia sp Mangifera indica L. Manihot esculenta Crantz Morinda lucida Benth. Musa paradisiaca L. Musa sapientum L. Ocimum gratissimum L Persea americana Mill. Picralima nitida (Stapf) Th & H. Dur. Psidium guajava L. Pteridium aquilinum Rauvolfia vomitoria Afzel. Maranthocloa Sp Schumanniophyton magnificum (R. Good). N. Hallé

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Family Asteraceae Asteraceae Mimosaceae Euphorbiaceae Apocynaceae Annonaceae Annonaceae Lauraceae Asteraceae Euphorbiaceae Solonaceae Caricaceae Chenopodiaceae Asteraceae Rutaceae Rutaceae Verbenaceae Rubiaceae Poaceae Burseraceae Asteraceae Arecaceae Annonaceae Myrtaceae Convolvulaceae Meliaceae Verbenaceae Anacardiaceae Euphorbiaceae Rubiaceae Musaceae Musaceae Lamiaceae Lauraceae Apocynaceae Myrtaceae Dennstaediaceae Apocynaceae Maranthaceae Loganiaceae

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Solonaceae Bignoniaceae Apocynaceae Mimosaceae Sterculiaceae Asteraceae Asteraceae Verbenaceae Apocynaceae Zingiberaceae

Solanum melongena L. Spathodea campanulata P. Beauv. Tabernaemontana crassa Benth. Tetrapleura tetraptera (Schum. & Thonn.) Taub. Theobroma cacao L. Tithonia diversifolia Gray Vernonia amygdalina Del. Vitex doniana Sweet Voacanga africana Stapf incl. Zingiber officinale Rosc.

Table 3: Citations of anti-malarial plant species in Andom village Scientific Name

Associated Plant

Plant Part

Ageratum conyzoides

associated with Dichrocephala Dichrocephala, Citrus limon Elaeis guineensis

Ageratum conyzoides Ageratum conyzoides Ageratum conyzoides Ageratum conyzoides Ageratum conyzoides

Administration

whole plant

Mode of preparation decoction

bath

Code_ Informant F2

fresh leaves

decoction

oral

F18

fresh leaves fresh leaves fresh leaves fresh leaves

grind maceration trituration trituration

rub on child oral friction press on painful side rub on body application on stomach oral

F24 F25 F3 F25

vaporation bath oral

F8 F16 F20 F26 F29 F1, F2, F4, F7, F9, F10, F12, F18, F19, F25, F28,M1, M2, M4 F4 F2, M2

Ageratum conyzoides Albizia adiantifolia

Voacanga

fresh leaves fresh leaves

trituration pound

Alchornea cordifolia

Rauvolfia vomitoria

fresh leaves

fresh leaves stem bark

warm on fire-frictionsqueeze decoction decoction

stem bark stem bark stem bark stem bark

decoction decoction decoction decoction

oral oral oral oral

Alstonia boonei Alstonia boonei

stem bark stem bark

decoction infusion

rectal oral

Alstonia boonei

stem bark

maceration

oral

stem bark fresh leaves fresh leaves stem bark

maceration decoction decoction decoction

rectal Vaporation bath oral oral

Alchornea cordifolia Alstonia boonei Alstonia boonei Alstonia boonei Alstonia boonei Alstonia boonei

Alstonia boonei Annona muricata Annona muricata Annonidium mannii

associated with Cymbopogon Associated with Vernonia Ctrus limon Enantia

associated with Carica associated with Coffea

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F10, F19 M6 F7

F10, F14, F18 F4 F16 F30 F1

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 10 | October 2013 | 692â&#x20AC;&#x201C;708 stem bark fresh leaves roots roots fresh leaves fresh leaves fruits

decoction decoction decoction decoction decoction decoction decoction

nasal oral auricular nasal oral Vaporation bath nasal

F8 F27 F8 F8 F28 F23 F8

fruits

maceration

rectal

F17

Capsicum frutescens Capsicum frutescens

Capsicum frutescens associated with Enantia associated with Spathodea associated with Spathodea Citrus limon, Theobroma Musa sapientum associated with Beilschmiedia associated with Clerodendrum associated with Coffea associated with Spathodea

fruits fresh leaves

oral nasal

F30 F3

Capsicum frutescens

associated with Spathodea

fresh leaves

Oral instillation

Capsicum frutescens Capsicum frutescens Capsicum frutescens Capsicum frutescens Carica papaya Carica papaya Carica papaya Carica papaya

associated with Spathodea associated with Spathodea Associated with Vernonia

fruits fruits fresh leaves fruits fresh leaves fresh leaves fresh leaves fresh leaves

decoction warm on fire-frictionsqueeze warm on fire-frictionsqueeze decoction decoction trituration decoction decoction decoction decoction decoction

auricular nasal rectal oral Vaporation bath Vaporation bath Vaporation bath Vaporation bath

F8 F8 F16 F11 F27 F18 F20 F16

fresh leaves

decoction

oral

F22

roots

decoction

oral

F22

fresh leaves fresh leaves seeds seeds whole plant

decoction maceration decoction maceration decoction

oral oral oral oral Bath

F4 F4 F4 F4 F2

fresh leaves

decoction

oral

associated with Coffea

fresh leaves

decoction

Vaporation bath

Musa sapientum, Thitonia diversifolia associated with Ageratum

fresh leaves

decoction

Vaporation bath

fruits

decoction

oral

F18

associated with Enantia chlorantha Associated with Alstonia associated with Carica associated with Carica's leaves associated with Carica's roots associated with Coffea Citrus limon

fruits

decoction

oral

fruits fresh leaves fruits

decoction decoction decoction

oral Vaporation bath oral

F26 F16 F22

fruits

decoction

oral

F22

fresh leaves fresh leaves

decoction decoction

Vaporation bath oral

F27 F28

Beilschmiedia sp Bidens pilosa Bridelia scleroneura Bridelia scleroneura Caffea robusta Caffea robusta Capsicum frutescens Capsicum frutescens

Carica papaya Carica papaya

Carica papaya Carica papaya Carica papaya Carica papaya Chenopodium ambrosioides Chenopodium ambrosioides Chromolaena odorata Chromolaena odorata Citrus limon Citrus limon Citrus limon Citrus limon Citrus limon Citrus limon Citrus limon Cofea robusta

associated with Coffea associated with Lippia Associated with Persea Citrus limon, Cymbopogon, Musa paradisiaca, Annona, Voacanga Psydium, Coffea, Eucalyptus, Citrus limon Psydium, Coffea, Eucalyptus, Citrus limon

associated with Dichrocephala

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 10 | October 2013 | 692â&#x20AC;&#x201C;708 Citrus limon Citrus limon Citrus reticula Clerodendrum splendens Clerodendrum splendens Clerodendrum splendens Clerodendrum splendens Clerodendrum splendens Clerodendrum splendens Coffea canephora Coffea canephora Coffea canephora Coffea canephora Coffea canephora Coffea canephora Coffea canephora Cymbopogon citratus Cymbopogon citratus Dacryodes edulis Dichrocephala integrifolia Dichrocephala integrifolia Dichrocephala integrifolia Dichrocephala integrifolia Dichrocephala integrifolia Dichrocephala integrifolia Dichrocephala integrifolia Dichrocephala integrifolia Dichrocephala integrifolia Dichrocephala integrifolia Elaeis guineensis Elaeis guineensis Elaeis guineensis Enantia chlorantha Enantia chlorantha

associated with Coffea associated with Lippia Associated with Persea Associated with Morinda

fruits fresh leaves fresh leaves fresh leaves

decoction decoction decoction trituration

oral Vaporation bath Vaporation bath oral

F28 F18 F20 M3

Associated with Vernonia

fresh leaves

trituration

rectal

F16

Capsicum frutescens

fresh leaves

maceration

rectal

F17

fresh leaves

trituration

oral

F16

fresh leaves

trituration

oral

F17

fresh leaves

trituration

oral

F27

fresh leaves

decoction

oral

F30

fresh leaves

decoction

oral

F22

fresh leaves

decoction

oral

F22

fresh leaves fresh leaves

decoction decoction

Vaporation bath Vaporation bath

F20 F27

fresh leaves fresh leaves fresh leaves roots stem bark whole plant

decoction decoction decoction decoction decoction decoction

Vaporation bath oral Vaporation bath oral oral Bath

F3 F15, F29 F16 F16 F18 F2

fresh leaves

decoction

oral

F18

fresh leaves

pound

Scarification

F16

fresh leaves

decoction

nasal

F26

fresh leaves

pound

F16

fresh leaves

pound

Application on stomach nasal

fresh leaves

pound

Scarification

fresh leaves

trituration

nasal

F18

fresh leaves

nasal

F3, F8, F10

roots

warm on fire-frictionsqueeze pound

Scarification

seeds sap fruits

oil palm wine oil

rub on child oral Massage

F24 F30 F6

stem bark stem bark

decoction decoction

oral oral

F29 F3

Annona, Persea, Capsicum, Elaeis associated with Carica's leaves associated with Carica's roots Associated with Persea Carica, Musa paradisiaca, Citrus limon Chromolaena, Psidium associated with Carica Zingiber, Alstonia associated with Lippia Ageratum, Musa paradisiaca, Chenopodium associated with Ageratum associated with Acmela

associated with Ageratum associated with Coffea associated with Tetrapleura Associated with Alstonia associated with Schumanniophyton

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F25

Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 10 | October 2013 | 692â&#x20AC;&#x201C;708 stem bark stem bark

decoction decoction

oral oral

M2 F27

Enantia chlorantha

stem bark

decoction

oral

F1, F2, F4, F7, F8, F9, F10, F14, F16, F18, F25, F28, M1, M4, M5

Enantia chlorantha

stem bark

maceration

oral

Enantia chlorantha Enantia chlorantha

Citrus limon Schumanniophyton, Picralima, Bidens

Eucalyptus camaldulensis Eucalyptus camaldulensis Ipomoea involucrata Ipomoea involucrata

associated with Carica's fresh leaves associated with Carica's roots associated with Lippia associated with Spathodea

fresh leaves

decoction

oral

F10, F16, F18 F22

fresh leaves

decoction

oral

F22

fresh leaves fresh leaves

Vaporation bath nasal

F18 F3

Ipomoea involucrata

associated with Spathodea

fresh leaves

decoction warm on fire-frictionsqueeze warm on fire-frictionsqueeze decoction decoction

Oral instillation

oral Vaporation bath

F1, M2 F18

oral Vaporation bath Scarification nasal

F15 F20 M6 F16

oral nasal nostril

M3 F23 F14

stem bark fresh leaves fresh leaves dead leaves

decoction decoction dry-squeeze warm on fire-frictionsqueez trituration ash warm on fire-frictionsqueeze decoction decoction decoction decoction

oral Vaporation bath Vaporation bath Bath

F14 F16 F27 F2

dead leaves

decoction

Vaporation bath

dead leaves fresh leaves

decoction decoction

Vaporation bath Vaporation bath

F23 F18

fresh leaves

oral

F13

fresh leaves fresh leaves

warm on fire-frictionsqueeze decoction decoction

oral Vaporation bath

F30 F20

stem bark stem bark

decoction decoction

oral oral

F27 F4

Khaya ivorensis Lippia sp

Lippia sp Mangifera indica Manihot esculenta Morinda lucida

Morinda lucida Morinda lucida Morinda lucida

Morinda lucida Musa paradisiaca Musa paradisiaca Musa paradisiaca

Musa sapientum Musa sapientum Ocimum gratissimum Ocimum gratissimum Persea americana Persea americana Picralima nitida Picralima nitida

Citrus limon, Ipomoea, Ocimum, Vitex, Carica, Dacryodes Associated with Persea Spathodea

Vernonia

associated with Carica associated with Coffea associated with Dichrocephala associated with Chromolaena associated with Coffea associated with Lippia

associated with Coffea Mangifera, Coffea, Citrus reticula, carica associated with Enantia

stem bark fresh leaves

fresh leaves fresh leaves Tuber fresh leaves

fresh leaves fresh leaves fresh leaves

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 10 | October 2013 | 692â&#x20AC;&#x201C;708 Psidium guajava Psidium guajava Psidium guajava Pteridium aquilinum Rauvolfia vomitoria Rauvolfia vomitoria Rauvolfia vomitoria Rauvolfia vomitoria

associated with Carica's roots associated with Carica's leaves associated with Coffea Sarcophrynium schweinfurthianum associated with Alchornea

fresh leaves

decoction

oral

F22

fresh leaves

decoction

oral

F22

fresh leaves fresh leaves

decoction decoction

Vaporation bath Vaporation bath

F3 F24

fresh leaves fresh leaves fresh leaves fresh leaves

decoction decoction decoction warm on fire-frictionsqueeze warm on fire-frictionsqueeze decoction pound

oral Massage oral Massage

F7 F16, F27 F15 F18

press on painful side

F25

decoction

oral nasal oral oral

Rauvolfia vomitoria

fresh leaves

Rauvolfia vomitoria Rauvolfia vomitoria Rauvolfia vomitoria Rauvolfia vomitoria

roots roots seeds stem bark

Sarcophrynium schweinfurthianum Schumanniophyton magnificum Schumanniophyton magnificum Schumanniophyton magnificum Schumanniophyton magnificum

Associated with Pteridium

fresh leaves

decoction

Vaporation bath

F3 F18 F26 F7, F18, F19, F25 F24

associated with Enantia

stem bark

decoction

oral

F27

Enantia

stem bark

decoction

oral

Solanum aethiopium

fresh leaves

pound

Scarification

F27

stem bark

decoction

oral

Solanum aethiopium

associated with Schumanniophyton Associated with Morinda

fresh leaves

pound

Scarification

F3, F4, F6, F9, F16, M6 F27

fresh leaves

oral

F16

stem bark

auricular

stem bark

decoction

nasal

Spathodea campanulata

Bridelia scleroneura, Tabernaemontana, Capsicum Bridelia scleroneura, Tabernaemontana, Capsicum Ipomoea involucrata, Capsicum frutescens

warm on fire-frictionsqueeze decoction

fresh leaves

nasal

Spathodea campanulata

Ipomoea involucrata, Capsicum frutescens

fresh leaves

Oral instillation

nasal

F27

Scarification auricular

F16 F8

nasal

Spathodea campanulata Spathodea campanulata Spathodea campanulata

Dichrocephala associated with Spathodea

fresh leaves stem bark

warm on fire-frictionsqueeze warm on fire-frictionsqueeze warm on fire-frictionsqueeze pound decoction

associated with Spathodea

stem bark

decoction

fresh leaves

Spathodea campanulata Acmella caulirhiza Tabernaemontana crassa Tabernaemontana crassa

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 10 | October 2013 | 692â&#x20AC;&#x201C;708 Tabernaemontana crassa

fresh leaves

Tabernaemontana crassa Tetrapleura tetraptera Cofea robusta Theobroma cacao Thitonia diversifolia Vernonia amygdalina Vernonia amygdalina Vernonia amygdalina Vernonia amygdalina Vernonia amygdalina Vitex doniana Sweet Voacanga africana Voacanga africana Voacanga africana Voacanga africana Voacanga africana Voacanga africana Voacanga africana Zingiber officinalis

press on painful side

F21

stem bark

warm on fire-frictionsqueeze decoction

oral

F13, F21

Elaeis guineensis

stem bark

rapure

Massage

Theobroma associated with Coffea associated with Chromolaena Alstonia

fresh leaves fresh leaves fresh leaves

decoction decoction decoction

oral oral Vaporation bath

F28 F28 F5

fresh leaves

decoction

oral

F20

Clerodendrum, Capsicum frutescens

fresh leaves

trituration

rectal

F16

fresh leaves

trituration

oral

F15

fresh leaves

trituration

oral

F26

roots

pound

nasal

F16, F27

associated with Lippia associated with Albizia

fresh leaves fresh leaves

decoction pound

F18 M6

associated with Carica

fresh leaves fresh leaves roots roots roots seeds roots

decoction decoction decoction decoction maceration

Vaporation bath Application on stomach Vaporation bath oral nasal oral nasal oral oral

associated with Cymbopogon

Characterization of recipes Recipes are characterized by the plant part, the pharmaceutical form, the mode of administration, and the degree of association of plant species involved. A total of nine plant parts were cited by Andom people for treating malaria, including: dead leaves, fresh leaves, roots, sap, seeds, stem barks, tubers, and fruits. Figure 1 illustrates the result. Fresh leaves (49% of citations) and stem barks (33%) are in this order the plant parts that are largely cited. Dead or dried leaves represent only 1.4% of citations. Sometimes, people of Andom village use the whole plant (1.4%). A total of eleven different mode of preparation of plants (or pharmaceutical forms)

decoction

F16 F15, F28 F6 F15 F6 F28 F16

were cited (figure 2): ash, decoction, drysqueeze, grind, infusion, maceration, oil, pounding, rapure, trituration, warm on firefriction-squeeze, and wine. Decoction (68% of citations) is the most important mode of preparation of anti-malarial plants. The relative importance of the modes of administration of recipes used as anti-malarial by Andom people is illustrated in figure 3. A total of 14 modes of administration are shown including: application on stomach, auricular, bath, friction, massage, nasal instillation, application on nostril, oral, pressing on painful side, rectal, rubbing on body, scarification, and vapour bath. Oral voice is largely cited (56%), followed by vapour bath (15%) and nasal instillation (11%). About 51.6% of the citations are made of combination of two, three, four, five, six, or seventh plant species.

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Global J Res. Med. Plants & Indigen. Med. | Volume 2, Issue 10 | October 2013 | 692â&#x20AC;&#x201C;708

Figure 1: Relative importance of plant parts cited for treating malaria in Andom village

Figure 2: Relative importance of the modes of preparation of recipes in the treatment of malaria in Andom village.

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Figure 3: Relative importance of modes of administration of recipes in the treatment of malaria in Andom village.

DISCUSSION Characteristics of recipes Recipes gathered in Andom village on antimalarial plants were compared to those obtained in the Dja Biosphere Reserve in the East Cameroon (Betti, 2001; 2003) and in the Ipassa-Makokou biosphere Reserve in Gabon (Betti et al., 2013), using almost the same method While leaves appear to be the most important plant parts used in Andom village and Ipassa-Makokou Biosphere Reserve (more than 50%), people living inside and in the periphery of the Dja Biosphere Reserve use mainly stem barks (60%) for treating malaria. Leaves arrive in the third position with only 11% of citations. Andom people have preferences in the use of freshly collected leaves (49%) than dried or dead leaves (1%). Studies had shown that there were quantitative and qualitative differences in the essential oil components of fresh and dry plant materials. Dry plant material might not be as potent as freshly collected materials (Idowu et al., 2010). As observed in the Dja and Ipassa-Makokou Biosphere Reserves, decoction is the main mode of preparation of recipes in Andom village. While people living in Andom village and the Dja Biosphere Reserve use mainly oral

voices, those living in the Ipassa-Makokou Biosphere Reserve in Gabon, prefer vaporation baths as the way of administration of recipes in the treatment of malaria. About half of the recipes indicated for treating malaria by people living in Andom are made of combination of many plant species. In the Ipassa-Makokou Biosphere Reserve, 73% of recipes were made of combination of many plant species. According to Rasoanaivo et al. (2011), there is evidence that crude plant extracts often have greater anti-plasmodial activity than isolated constituents at an equivalent dose. Use of medicinal plants out of Andom village Citations of plants used in Andom village were compared to those mentioned in African countries. Table 4 presents each plant species cited in Andom, the countries where the same plants are indicated with the references in brackets. A total of 29 plants (57%) used by people living in Andom village as anti-malarial are also known in other region of Cameroon and other African countries for the same usage. The most cited plant species are: Alstonia boonei (8 countries), Rauvolfia vomitoria (7), Carica papaya (6), Cymbopogon citratus (5), Morinda lucida (5), and Mangifera indica (5), Enantia chlorantha (4), Picralima nitida (4).

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Table 4: Use of anti-malarial plants out of the Andom village Sources 1 : Adjanohoun et al. (1996) ; 2 : Bitsindou (1996) ; 3 : Diafouka (1997) ; 4 : Iwu et al. (1992) ; 5 : Magilu et al. (1996) ; Ngalamulume et al. (1995) ; 7 : Richel (1995) ; 8 : Cousteix (1961) ; 9 : Dijk (1999) ; 10 : Betti (2001) ; 11 : Iwu (1994) ; 12 : Betti (2003), 13 : Tchouamo and Njoukam (2000) ; 14 : Betti and Van Essche (2001); 15: Satoing et al. (2011); 16 : Betti (2002) ; 17: Idowu et al. (2010); 18: Betti et al. (2013) .

Plant species Acmella caulirhiza Ageratum conizoides Alstonia boonei Annona muricata Bidens pilosa Capsicum frutescens Carica papaya Citrus limon Chromolaena odorata Clerodendrum splendens Cymbopogon citratus Dacryodes edulis Elaeis guineensis Enantia chlorantha Ipomoea involucrata Mangifera indica Manihot esculenta Morinda lucida Musa paradisiaca Ocimum gratissimum Persea americana Picralima nitida Psidium guajava Rauvolfia vomitoria Schumanniophyton magnificum Spathodea campanulata Tabernae montana crassa Tetrapleura tetraptera Vernonia amygdalina

Countries (reference) Cam (9, 10) Gab (18) Cam (1, 8, 9, 10,12,14, 16) ; Cng (3) ; Ga (18); Geq (2) ; Nig (7, 17), Sén (7) ; DRC (5) ; Tog (7) Gab (18) Cam (1, 10, 12) ; DRC (2) Cam (10, 12, 14, 15) ; DRC (2), Cng (3), Gab (18) Cam (1, 10, 12, 13, 14, 15) ; Cng (2, 3) ; Nig (7, 17), Gha (15), Tog (7); Gab (18) Cam (9, 10, 12, 14, 15) ; DRC (2, 5), Gab (18) Gab (18) Geq (2) ; Cng (2) ; Gab (2, 18) Cam (1, 2, 9, 10, 12, 14, 15) ; Cng (2, 3) ; DRC (2); Ni (17); Gab (18) Gab (18) Cam (10, 12, 14) ; DRC (2); Gab (18) Cam (8, 9, 10, 12, 14, 16) ; Geq (2) ; Cng (3); Gab (18) Gab (18) Cam (10, 12, 14, 15); Gab (2, 18) ; DRC (2) ; Cg (3), Ni (17) Gab (18) Cam (9, 10, 12, 14, 16) ; Cng (2) ; DRC (5) ; Nig (7, 11, 17), Tog (7) Gab (18) Cam (10, 12, 14) ; Cng (3) ; Ni (17) Gab (18) Cam (1, 9, 10, 12, 14, 16) ; DRC (5), Nig (4, 11), Gab (18) Cam (15), Ni (17), Gab (18) Cam (1, 9, 10, 12, 14) ; Gab, RCA (2) ; DRC (2, 5, 6) ; Nig (7, 17) ; Tog (7) ; Bén (7) Cam (10, 12, 14) Cam (1, 9, 10, 12, 14) ; Cng (2) Cam (9) Cam (10, 12, 14) Cam (10, 15), Ni (17), Gab (18)

Countries: Ben. : Benin ; Cam : Cameroon ; Cng : Congo Brazzaville ; Gha : Ghana ; Geq : Equatorial Guinea ; Nig : Nigeria ; Sen : Senegal ; Gab : Gabon ; RCA : Central African Republic; DRC : Democratic Republic of Congo ; Tog : Togo.

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Eight out of the twenty nine plant species (27.6%) also known for their anti-malarial usage out of Andom village are well recognized for their real activity against malaria including: Alstonia boonei, Carica papaya, Citrus limon, Cymbopogon citratus, Enantia chlorantha, Morinda lucida, Picralima nitida, and Vernonia amygdalina. Alstonia boonei, Carica papaya, Citrus limon, Cymbopogon citratus, Enantia chlorantha, Picralima nitida and Vernonia amygdalina have been reported to be active against Plasmodium spp (Betti, 2001; 2003; Betti et al., 2013). Clinical investigation of Carica papaya, Cymbopogon citratus, Ocimum gratissimum, and Vernonia amygdalina, used as traditional medicines in Kinshasa, the Democratic Republic of Congo, to treat malaria patients showed significant removal of parasites in the blood, as well as elimination of clinical detection of disease (Taba et al., 2012). The anti-malarial activity of Morinda lucida (Rubiaceae) has been established on Plasmodium berghei (Makinde and Obih, 1985; Obih et al., 1985), P. yoelii nigeriensis (Agomo et al., 1992) and P. falciparum (Gbeassor et al., 1988; Koumaglo et al., 1992; Sittie et al., 1999; Tona et al., 1999). A prophylactic activity has also been established by Makinde and Salako (1991). According to Koumaglo et al. (1992), this activity is due to the presence of three compounds (anthraquinones) including digitolutein, rubiadin-1-methyl ether and damnacanthal isolated from the stem and root barks. Tona et al. (1999) having put in evidence Morinda’s activity on leaves which

do not contain the above compounds, concluded that the leaves’ activity may come from other type of compounds. The age of development of the plant part does not have any effect on the activity of Morinda (Tona et al. 1999). Iwu (1994) revealed that the antimalarial activity of M. lucida is largely exploited in primary health centers in Nigeria. However studies have reported the toxicity of that plant species (Idowu et al., 2010). CONCLUSION The fact that some plant species cited by Andom people be recognized for their activity against Plasmodium, is a credibility index which can be attributed to the pharmacopoeia of those people. This also illustrates the efficiency of the method used to identify medicinal plants of the Andom village. The glaring development challenge at the background of what precedes is the pressing need to implement strategies and programmes to identify active chemical substances of other plant species of this list, which have not yet been investigated for their chemical and antimalarial activities. ACKNOWLEDGEMENTS We thank all the villagers who collaborated with us in this study. This paper has been produced with the financial assistance of the Cameroon Government and the Japanese Cooperation (JICA) under the FOSAS Forest-Savanna Sustainability Project, Cameroon”.

REFERENCES Adjanohoun E, Aboubakar N, Dramane K, Ebot ME, Ekpere JA, Enow-Orock EG, Focho D, Gbilé ZO, Kamanyi A, Kamsu Kom J, Keita A, Mbenkum T, Mbi CN, Mbiele AL, Mbome IL, Mubiru NK, Nancy WL, Nkongmeneck B, Satabié B, Sofowora A, Tamze V, Wirmum, CK (1996). Contribution to Ethnobotanical and Floristic Studies in Cameroon. CSTR/OUA.

Agomo PU, Idigo JC, Afolabi BM (1992). Antimalarial medicinal plants and their impact on cell populations in various organs of mice. African journal of medicine and medical sciences. 21 (2):39–46.

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Balick MJ (1985). Useful plants of Amazonia: a resource of global importance In G.T. Prance & T.E. Lovejoy, (eds). Key environments: Amazonia. Oxford, Pergamon Press. Balick MJ (1990). Ethnobotany and the identification of therapeutic agents from the rainforest. In. Chadwick DJ & Marsh J, (eds). Bioactive compounds from plants (Ciba Foundation Symposium No. 154). Wiley, Chichester: 22–32. Balick

MJ (1994). Ethnobotany, drug development and biodiversity conservation – exploring the linkages. In. Chadwick DJ & Marsh J, (eds). Ethnobotany and the Search for New Drugs (Ciba Foundation Symposium No. 185). Wiley, Chichester: 4–18.

Betti JL (2001). Usages traditionnels et vulnérabilité des plantes médicinales dans la réserve de biosphère du Dja, Cameroun. Thèse de Doctorat, Université Libre de Bruxelles. Betti JL (2002). Medicinal plants sold in Yaoundé markets, Cameroon. African Study Monographs. 23 (2): 47–64. Betti JL (2003). Plantes utilisées pour soigner le paludisme dans la Réserve du Dja, Cameroun. Revue de Médecines et Pharmacopées Africaines. 17: 121–130. Betti JL, Van Essche K (2001). Enquêtes sur la pharmacopée populaire et spécialisée dans la réserve de faune du Dja (Cameroun): premiers résultats sur les plantes utilisées pour taiter la fièvre ou le paludisme en pharmacopée populaire. Etnofarmacologia. 1: 46–62.

Betti JL, Midoko Iponda D.Yongo OG, Obiang Mbomio D, Mikolo Yobo C, Ngoye A, Issembe Y (2013). Ethnobotanical study of medicinal plants of the IpassaMakokou Biosphere Reserve, Gabon: plants used for treating malaria. Journal of medicinal plants research. 7: 2300– 2318. Bitsindou M (1996). Enquêtes sur la phytothérapie traditionnelle à Kindamba et Odzala. Thèse de Doctorat Université Libre de Bruxelles. Cotton (1996) Ethnobotany. Principles and applications. Ed. Wiley, 424p Cousteix P-J (1961). L'art et la pharmacopée des guérisseurs Ewondo (Région de Yaoundé). Recherches et études camerounaises, Yaoundé, IRCAM, 1961 : 86 p. Diafouka A (1997). Analyse des usages des plantes médicinales dans quatre régions du Congo – Brazzaville. Thèse de Doctorat Université Libre de Bruxelles. Dijk J F W (1999). Non-timber forest products in the Bipindi-Akom II region, Cameroon. A socio-economic and ecological assessment. The TropenbosCameroon programme. Gbeassor M, Kossou Y, De Souza C, Amegbo K, Denke A (1988). Action de quelques plantes médicinales sur la croissance du Plasmodium falciparum in vitro. Bulletin de Médecine Traditionnelle et Pharmacopées. 4 (2): 139–146. Idowu O A, Soniran O T, Ajana O, Aworinde D O (2010). Ethnobotanical survey of antimalarial plants used in Ogun State, Southwest Nigeria. African Journal of Pharmacy and Pharmacology. 4 (2): 055–060.

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Iwu M M (1994). African medicinal plant in the search for new drugs based on ethnobotanical leads. In. Chadwick DJ & Marsh J (eds) Ethnobotany and the Search for New Drugs (Ciba Foundation Symposium No. 185). Wiley, Chichester: pp. 116–129. Iwu M M, Klayman D L (1992). Evaluation of the in vitro antimalarial activity of Picralima extracts. Journal of Ethnopharmacology, 36 (2): 133–135. Iwu M M, Jackson JE, Tally JD, Klayman DL (1992). Evaluation of plant extracts for antileishmanial activity using a mechanism-based radiorespirometric microtechnique (RAM): Planta Medica 58 (1992): 436–441. King SR, Tempesta MS (1994). From shaman to human clinical trials: the role of industry in ethnobotany, conservation and community reciprocity. In Chadwick DJ & Marsh J (eds) Ethnobotany and the Search for New Drugs (Ciba Foundation Symposium No. 185). Wiley, Chichester: 197-206. Koumaglo K, Gbeassor M, Nikabu O, de Souza C, Werner W (1992). Effects of three compounds extracted from Morinda lucida on Plasmodium falciparum. Planta Medica 58 (6): 533–534. Magilu M, Mbuyi M, Ndjélé MB (1996). Plantes médicinales utilisées par les pygmées (Mbute) pour combattre le paludisme dans la zone de Mambasa, Ituri, Zaïre. In L.J.G. van der Maesen, X.M. van der Burgt & J.M. van Medenbach de Rooy (eds.) Kluwer Academic publishers. The Netherlands. The biodiversity of African Plants, 741– 746.

Makinde JM, Obih PO (1985). Screening of Morinda lucida leaf extract for antimalarial action on Plasmodium berghei berghei in mice. African journal of medicine and medical sciences, 14 (12): 59–63. Makinde JM, Salako LA (1991). The antimalarial activity of some Nigerian medicinal plants on Plasmodium berghei berghei. Quatrième symposium inter-africain OUA/CSTR sur la pharmacopée traditionnelle et les plantes médicinales africaines: rapport et recommandations. Abuja Nigeria 1822 juillet 1988: 424–425 . Ngalamulume Tschimuene J, Paulus SJ, Kabeya M, Nlandus L., Kizika K (1995) Plantes médicinales à usage domestique cultivées dans deux quartiers de Kinshasa. Bull. Méd. Trad. Pharm., 9, (2): 9–14. Obih PO, Makinde M, Laoye OY (1985). Investigation of various extracts of Morinda lucida for antimalarial action on Plasmodium berghei berghei in mice. African journal of medicine and medical sciences. 14 (12): 45–49. Oketch-Rabah HA, Mwangi JW (1998). La médecine traditionnelle et les plantes médicinales ont-elles une place dans la lutte antipaludique? 3ème Conférence panafricaine sur le paludisme. Naïrobi/Kénya, 22-24 juin 1998. http:// www.chez.com/malaria/09fran 15.htm. Richel T (1995). Les plantes médicinales d'Afrique occidentale. Essai de synthèse sur base de la banque de données pharmel. Thèse Doctorat Université Libre de Bruxelles.

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Rasoanaivo1 P, Wright CW, Willcox ML, Gilbert B (2011). Whole plant extracts versus single compounds for the treatment of malaria: synergy and positive interactions. Malaria Journal 2011, 10 (Suppl 1). http://www.malariajournal.com/content/ 10/S1/S4. Saotoing P, VroumsiaToua, Tchobsala, Tchuenguem Fohouo F-N, Njan Nloga A-M, Messi J (2011). Medicinal plants used in traditional treatment of malaria in Cameroon. International Journal of the Physical Sciences, 3 (3): 104–117. Sittie AA, Lemmich E, Olsen CE., Hviid L, Kharazmi A, Nkrumah FK, Christensen SB (1999) Structure activity studies: in vitro antileishmanial and antimalarial activities of anthraquinones from Morinda lucida. Planta Med., 65(3): 259–261.

Source of Support: Government of Cameroon and the Japanese Cooperation (JICA) under the FOSAS Forest-Savanna Sustainability Project, Cameroon

Taba KM, Paulius J, Kayembe JS (2012). Malaria: Novel plant remedies show great promises in treating the deadly disease. Global J Res. Med. Plants & Indigen. Med., Volume 1 (3): 62–68. Tchouamo IR, Njoukam R (2000) Etude de quelques ligneux utilises en médecine traditionnelle par les Bamileke des Hauts-Plateaux de l’Ouest du Cameroun. Ethnopharmacologia. 26: 14–22. Tona L, Ngimbi NP, Tsakala M, Mesia K, Cimanga K, Apers S, De Bruyne T., Pieters L, Totté J, Vlietinck A.J. (1999). Antimalarial activity of 20 crude extracts from nine African medicinal plants used in Kinshasa, Congo. Journal of Ethnopharmacology 68: 193–203. World Malaria Organization Report 2008, Geneva, World Health Organization, 2008

Conflict of Interest: None Declared

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Research article STANDARDIZATION OF ERANDAMOOLADI KWATHA CHURNA – A COMPOUND FORMULATION USED IN MEDICATED ENEMA THERAPY (BASTI KARMA) Lohith B A1*, Sunil Kumar K N2, Girish K J3 1

Associate professor, Department of Panchakarma, Sri Dharmasthala Manjunatheshwara College of Ayurveda and Hospital, Tanniruhalla, BM Road, Hassan – 573201, Karnataka, India 2 Sri Dharmasthala Manjunatheshwara Centre for Research in Ayurveda and Allied sciences, Lakshminarayana Nagar, Kuthpady, Udupi - 574118, Karnataka, India 3 Professor, PG Department of Panchakarma, Sri Dharmasthala Manjunatheshwara College of Ayurveda and Hospital, Tanniruhalla, BM Road, Hassan – 573201, Karnataka, India *Corresponding Author: Mobile: +919886749168; E-mail address: drlohithpk@gmail.com

Received: 26/08/2013; Revised: 27/09/2013; Accepted: 04/10/2013

ABSTRACT To assure therapeutic efficacy and safety, the standardization of Ayurvedic compound plays an important role. Erandamooladi Kwatha Churna is a poly herbal formulation widely used in Ayurveda clinical practice with multi fold benefits like Agni Deepana (improving digestive fire), Ama Pachaka (digestion of undigested material) Sroto Shodhana (cleansing of micro channels) specifically to management of Gridhrasi (Sciatica). There are no work on the standardisation aspect of this formulation though individual herbs used for the preparation has been studied. This study highlights physico-chemical characterization, HPTLC and densitogram profile of Erandamooladi Kwatha Churna which can be applied for authentication of this poly herbal formulation. Formulation were prepared by combining all the drugs and subjected for detailed physico-chemical and HPTLC analyses. The results obtained are considered as tools for assistance to the regulatory authorities and manufacturers for developing standard formulation aiming for great efficacy. KEY WORDS: Erandamooladi Kwatha Churna, sciatica, poly herbal formulation, high performance thin layer chromatography, standardization

Cite this article: Lohith. B. A., Sunil Kumar. K. N., Girish. K. J., (2013), STANDARDIZATION OF ERANDAMOOLADI KWATHA CHURNA – A COMPOUND FORMULATION USED IN MEDICATED ENEMA THERAPY (BASTI KARMA), Global J Res. Med. Plants & Indigen. Med., Volume 2(10): 709–715

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INTRODUCTION Ayurveda, Indian system of medicine is the first recorded medical science. In recent years there is global revolution worldwide towards acceptance of this holistic science owing to its effectiveness and safety. The increasing demand has created great need to standardize herbal medicines for scientific base of acceptance. The earliest references of drug standardizations are mentioned in Ayurveda classics under the specialty of Bhaishajya Kalpana and Rasa Shastra which exclusively deal with drug formulation and manufacturing. Hence standardization and development of reliable quality protocols are important (Anantanarayana DB, 2002). The methods though crude but specify the required standards of the raw drugs as well as best quality of Ayurveda medicine. Sharngadhara, who pioneered Ayurvedic pharmacy has given best qualities of medicine along with the methodology of preparation of specified formulation such as Gutika (tablets),

Ghrita (medicated ghee), Taila (medicated oils) Avaleha (medicated elixirs), (Parashurama Shastri, 2000), and so on. In fact, it constitutes the first ever described good manufacturing practice and standard methods of quality control. Hence standardization and development of reliable quality protocols are important. Plant material when used in bulk quantity may vary in its chemical content and therefore, in its therapeutic effect according to different batches of collection. It may depend on the collection in different season and/or collection from sites with different environmental surrounding or geographical location. The increasing demand and persisting stage, authentic raw materials have made it incumbent, to maintain uniformity in the manufacture of Ayurvedic medicines so as to promise the quality control and quality assurance (WHO, 1992). Various formulations are described in Ayurvedic texts to treat Gridhrasi (sciatica). Erandamuladi Niruha Basti (medicated enema) is one among them.

Table 1. The Erandamooladi Kwatha Churna (Trikamji Yadavji, 2009) Sanskrit name Eranda Palasha Rasna Ashwagandha Atibala Guduchi Punarnava Aragvadha Devadaru Madhanaphala Shatahva Hapusha Priyangu Pippali Madhuka Bala Vatsaka Musta

Botanical name Ricinus comunis Butea monosperma Pluchea lanceolata Withania somnifera Abutilon indicum Tinospora cordifolia Boerhvavia diffusa Cassia fistula Cedrus deodara Randia spinosa Anethum sowa Juniperus communis Callicarpa macrophylla Piper longum Glycyrrhiza glabra Sida cordifolia Holarrhena antidysentrica Cyperus rotandus

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Considering therapeutic utility of the Erandamooladi Niruha Basti, a thought was given to standardize the same for multiple usage as Agni Deepana (improving digestive fire), Ama pachaka (digestion of undigested material) Sroto Shodhana (cleansing of micro channels of the body) and specifically to management of Gridhrasi (Sciatica). Development of a composite standardization protocol for Erandamooladi Niruha Basti was aimed in this study. The combination of the drugs mentioned in Table 1 are used to prepare the Kashaya (decoction) to be used for the administration of Niruha Basti (rectal route administration of medicine) with a wide range of applications on different conditions. Most of the drugs used in this formulation possess the properties like Ushna Veerya (hot in potency), Laghu (light), Ruksha (dryness) Gunas and does Deepana (digestive) and Lekhana Karma (scraping effect). It is indicated in pain in Janga (knee), Uru (leg region), Pada (foot) and Prusta (low back region and in Kapha-avrutha (channels obstructed by phlegm) conditions. The Erandamooladi formula acts as a Maruthanigrahana (controls movements), in case of Mala-mutra Sanga (obstruction to fecal and urine), Arsha (piles) Anaha (flatulence) and Admana (distention of abdomen) (Trikamji Yadavji, 2009). MATERIALS AND METHODS Instrumentation and techniques: High performance thin layer chromatography (HPTLC) studies were done at SDM Centre for Research in Ayurveda and Allied Sciences, Kuthpady, Udupi, Karnataka, India as per standard procedure (I Stahl, 1969; PD Sethi, 1996; Khandelwal KR, 2005). Plant materials: Required plant medicines were collected from authorized raw drugs suppliers of Chaitahanya Pharmaceuticals Bellary, Karnataka, India. The raw materials were first identified and authenticated by a team of botanists at Chaitahanya Pharmaceuticals, Bellary, Karnataka, India

Preparation of Erandamooladi Kwatha churna (EKC): As per the textual description (Parashurama, 2000) and guidelines in Ayurvedic formulary of India (AFI, 2003) the all drugs described above were powdered separately and mixed equally (Trikamji Yadavji, 2009). 1kg of final output of powder was obtained after 10% of loss during processing. All the sample powders were passed through 80 mesh size. Instrumentation (Anonymous, 2003).

and

techniques

1. Loss on drying at 105oC: 10 g of sample was placed in tared evaporating dish. It was dried at 105˚C for 5 hours in hot air oven and weighed. The drying was continued until difference between two successive weights was not more than 0.01 after placing in desiccator. Percentage of moisture was calculated with reference to weight of the sample. 2. Total Ash: 2 g of sample was incinerated in a tared platinum crucible at temperature not exceeding 450˚C until carbon free ash is obtained. Percentage of ash was calculated with reference to weight of the sample. 3. Acid insoluble Ash: To the crucible containing total ash, add 25ml of dilute HCl. Collect the insoluble matter on ashless filter paper (Whatman 41) and wash with hot water until the filtrate is neutral. Transfer the filter paper containing the insoluble matter to the original crucible, dry on a hot plate and ignite to constant weight. Allow the residue to cool in suitable desiccator for 30 minutes and weigh without delay. Calculate the content of acid insoluble ash with reference to the air dried drug. 4. Alcohol soluble extractive: Weigh accurately 4 g of the sample in a glass stoppered flask. Add 100 ml of distilled Alcohol (approximately 95%). Shake occasionally for 6 hours. Allow to stand for 18 hours. Filter rapidly taking care not to lose any solvent. Pipette out 25ml of the

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filtrate in a pre-weighed 100 ml beaker. Evaporate to dryness on a water bath. Keep it in an air oven at 105C for 6 hours, cool in a desiccator for 30 minutes and weigh. Calculate the percentage of Alcohol extractable matter of the sample. Repeat the experiment twice, and take the average value. 5. Water soluble extractive: Weigh accurately 4 g of the sample in a glass stoppered flask. Add 100 ml of distilled water, shake occasionally for 6 hours. Allow to stand for 18 hours. Filter rapidly taking care not to lose any solvent. Pipette out 25ml of the filtrate in a pre-weighed 100 ml beaker. Evaporate to dryness on a water bath. Keep it in an air oven at 105C for 6 hours. Cool in a desiccator and weigh. Repeat the experiment twice. Take the average value. 6. High Performance Thin Layer Chromatography (HPTLC): 1g of powder was extracted with 20 ml of alcohol with successive method (Lala, 1993) 15 and 30 micro liter µl of the above extract was applied on a precoated silica gel F254 on aluminum plates to a band width of 8 mm using Linomat 5 TLC applicator. The plate was developed in Toluene: Ethyl acetate: Formic acid (6: 2: 1.8). The developed plates were visualized in UV 254, 366 and after derivatisation with vanillin-sulphuric acid and scanned under UV 254, 366, and at 540 nm. Rf values, colour of the spots and densitometric scan were recorded.

RESULTS & DISCUSSION: The results of the Physico-chemical parameters of EKC has been tabulated in Table 2. Thin layer chromatography (TLC): TLC fingerprint profile is a systematic representation of all the constitution of samples resolved in the given chromatographic system. TLC photo documentation of EKC is presented in Figure 1. High Performance Thin Chromatography (HPTLC):

Layer

HPTLC fingerprint of butanol soluble portion of EKC has been developed. The purity of the band in the sample extracts was confirmed by comparing the absorption spectra recorded at start, middle, and end positions of the band. The video densitometric images of chromatoplate are depicted. HPTLC densitometric scan at UV 254, 366, 620 nm are presented in Figure 2. The Rf values are tabulated in Table 3. Rf values of the spots and their colour by TLC photo-documentation of EKC extracts have been developed. Chloroform extract of EKC at 254 nm showed 10 spots (0.12 Green, 0.18 Green, 0.32 D green, 0.39 Green, 0.43 Green, 0.53 Green, 0.57 Green, 62 Green, -0.69 Green, 0.82 D green) whereas under 366 nm it showed 5 spots (0.29 F.Blue, 0.43 F. Green, 0.48 F.Blue, 0.62 F.Blue, 0.65 F.Blue, 0.72 F.Green, 0.78 F.Pink, 0.91 F.Blue) and 8 spots (0.12 Violet, 0.18 Violet, 0.32 Yellowish brown, 0.39 Blue, 0.51 Blue, 0.53 Pink, 0.65 Blue, 0.69 Pink, 0.76 D blue, 0.82 D blue) after derivatisation using toluene: methyl acetate (6.5:2.5) as solvent system.

Table 2: Physico-chemical parameters of Erandamooladi Kwatha Churna Parameter

Result (n = 3; % w/w)

Loss on drying at 105oC Total ash Acid insoluble ash Water soluble extractive Alcohol soluble extractive

10.99 6.53 1.44 13.4 5.6

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Figure 1. TLC photodocumentation of alcohol extract of Erandamooladi Kwatha Churna

At UV 254 nm

At UV 366 nm

Post- derivatisation with vanillinSulphuric acid

Track 1- 15 µl and Track 2 - 30 µl Solvent system - Toluene : Ethyl acetate: Formic acid (6:2:1.8)

Figure 2. HPTLC Densitometric scan of alcohol extract of Erandamooladi Kwatha Churna

B A- 15 µl – 254 nm and B - 15 µl – 366 nm Solvent system - Toluene : Ethyl acetate: Formic acid (6:2:1.8

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Table 3. Rf value of Erandamooladi Kwatha Churna Alcohol extract (30 µl) At UV 254 nm 0.12 Green 0.18 Green 0.32 D green 0.39 Green 0.43 Green 0.53 Green 0.57 Green 0.62 Green 0.69 Green 0.82 D green -

At UV 366 nm

Post - derivatisation

0.12 Violet 0.18 Violet 0.29 F.Blue 0.32 Yellowish brown 0.39 Blue 0.43 F. Green 0.48 F.Blue 0.51 Blue 0.53 Pink 0.62 F.Blue 0.65 F.Blue 0.65 Blue 0.69 Pink 0.72 F.Green 0.76 D blue 0.78 F.Pink 0.82 D blue 0.91 F.Blue F – Flourescent; D – Dark

CONCLUSION Despite the advent of modern technology in standardization of compound formulations, only a few Ayurvedic poly herbal medicines have been standardized so for. This study was aimed at authentication of ingredients used in the sample and chemical characterization using advanced methodology. Considering its wide range of age, EKC was selected for study. Physico-chemical standardization of EKC was carried out. Individual ingredients of the formulation were authenticated and standardized as per WHO criteria on herbal pharmacopeia. TLC photo documentation of EKC with its ingredients was carried out. HPTLC fingerprint of chloroform and alcohol extract (successive) of EKC was developed. The product EKC was analyzed for its fingerprint in comparison with its ingredients. The current investigation can be used as

standardization test for this compound formulation. Further, detailed macro & microscopic examination of the raw drug individually and powdered form would add to the standardization test of EKC. ACKNOWLEDGEMENT Authors are highly grateful to our revered President, Dr. D. Veerendra Heggade and Dr. B. Yashoverma, Secretary, SDM Educational Society, Ujire, Karnataka, India for their encouragement. Authors greatly regard the constant support of Dr. Prasanna N Rao, Principal, SDM College of Ayurveda & Hospital, Hassan, Karnataka, India and Dr. B. Ravishankar, Director, SDM Centre for Research in Ayurveda and Allied Sciences, Udupi, Karnataka, India for providing the facilities and for their help in carrying out the studies.

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REFERENCES Anantanarayana, D.B. (2002). Proceeding of International Congress on Ayurveda, 28–30th January. 2002; 67. Anonymous (2003). The Ayurvedic Formulary of India. New Delhi: Government of India, Ministry of Health and family welfare. Part-I, 2nd ed. I Stahl (1969). Thin Layer Chromatography, A Laboratory Hand Book. Berlin: Springer-Verlag. 52–86. Khandelwal KR (2005). Practical Pharmacognosy:. Techniques & Experiments, Pune: Nirali Prakashan. 13th ed., 147–155. Lala

PB (1993). Lab manuals of Pharmacognosy, Calcutta: CSI publishers & distributors. 5th ed.

Source of Support: Nil

Sethi PD (1996). High Performance Thin Layer Chromatography, New Delhi: CBS Publishers and Distributors, 1st ed., 1– 56. Shastri Parashurama (2000). Sharngadhara Samhita with Deepika and Gudhartha Deepika of Adhamalla and KashiramaVaidya: New Delhi: Chaukambha Orientalia, 214. WHO (1992) Organization Mondiale De La Sante, Quality control methods for medicinal plant materials, (World Health Organization, 559, rev.1, Original English, 1992), 159. Yadavji Trikamji (1994). Charaka Samhita with Ayurveda Dipika commentary of Chakrapanidatta, 5th edition. Varanasi: Chaukhambha Surabharathi Prakashana; 696.

Conflict of Interest: None Declared

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Research article ROLE OF PHALAGHRITA AND UTTARBASTI IN THE MANAGEMENT OF VANDHYATVA (INFERTILITY) WITH REFERENCE TO CERVICAL FACTOR Pandya Neha R1, Donga Shilpa B2*, Mistry I U3 1

Reader, Department of Streeroga & Prasutitantra, Sheth J.P. Government Ayurveda College, Bhavnagar, Gujarat, India. 2 Associate professor, Dept. of Streeroga & Prasutitantra, I.P.G.T. & R.A., Gujarat Ayurved University, Jamnagar, Gujarat, India. . 3 Ex. Professor & Head of Department, Dept. of Kaumarbhritya, I.P.G.T. & R.A., Gujarat Ayurved University, Jamnagar, Gujarat, India. *Corresponding Author: E-Mail: drshilpadonga@yahoo.com; Mobile No: +919825646796

Received: 13/07/2013; Revised: 27/09/2013; Accepted: 01/10/2013

ABSTRACT Vandhyatva (infertility) has been a long standing problem since ancient times. Many herbal and herbo – mineral formulations are mentioned as a treatment of infertility in the ancient texts, but they are not categorized according to the responsible factor of infertility. It is the need of the hour to evaluate the efficacy of formulations with respect to various factors of infertility. With the above aim a clinical study was conducted to evaluate the efficacy of Phalaghrita and Uttarbasti on cervical factor i.e. scanty cervical mucus. For clinical trial total 13 patients were selected and randomly divided into two groups. In Group A, 15 ml of Phalaghrita was given orally twice a day for 2 months with warm Milk and in Group B; Intracervical Uttarbasti of Phalaghrita (5 ml) was administered in every sitting for 6 days with 3 days interval after cessation of menses subsequently for two cycles. Sim’s hunter and Moghissi’ cervical mucus Test and Post coital test were selected for the diagnosis and for evaluation of efficacy of therapy on cervical factor. Statistically significant result were found on spinnbarkeit (p< 0.001), density of sperm (p<0.05), cellularity (p 0.01) and ferning of cervical mucus (p 0.001)in group A, and statistically significant (p 0.001, 68%) result was found on amount of cervical mucus and motility of sperm (p<0.01) in Group B. Hence, in nutshell it was concluded that oral use of Phalaghrita showed better results in comparison to use as an Uttarbasti. KEYWORDS: Vandhyatva (infertility), cervical factor (scanty cervical mucus), Phalaghrita, Intra cervical Uttarbasti

Cite this article: Pandya Neha. R., Donga Shilpa. B., Mistry. I. U.,(2013), PHYTO-CHEMISTRY, ANTIBACTERIAL ACTIVITY AND CHROMOSOME NUMBER OF CENTAUREA SOLSTITIALIS L. GROWN IN ALGERIA, Global J Res. Med. Plants & Indigen. Med., Volume 2(10): 716–723

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INTRODUCTION: In the present scenario of the rapid advancement in technology, infertility is still a problem that has continued since ages. Many factors are responsible for female infertility, which is Tubal factor – 45%, ovarian factor – 25%, cervical factor - 20% and uterine factor10% (D. C. Dutta, 2009). J. M. Sims, (1968) first identified the influence of cervical factor in causing infertility. The cervical mucus acts as a filter allowing only functioning best spermatozoa to penetrate cervical mucus. When this is hostile and unfavorable it reduces the quality or quantity of sperm, affect sperm viability and ultimately fertility. Acharya Sushruta (Ambika Dutt Shastri, 2007) has described the essential factors for conception i.e. Ritu (season or ovulatory period), Kshetra (field i.e. reproductive organs), Beeja (seed i.e. ovum/sperm) and Ambu (water i.e. nutritive material & hormones) which are responsible to maintain the pregnancy and defect in either of these factors may result into infertility. Less quantity and poor quality of cervical mucus may be due to inadequate oestrogen level or less utilization of estrogen through receptor is the main factor of infertility caused by cervical factor. Proper secretion of cervical mucus is a resultant of balanced Tridosha (three humors of body), Prasada Rasa dhatu(essence plasma or nutrients), Rakta dhatu (Blood), Mamsa dhatu (Muscles, tissues) and Sthanika Agni (local metabolism) (at cellular level) according to Ayurveda. Functions of cervical mucus can be linked with the Kledana Karma (moisturizing action) of Kapha. Vata vitiated due to Ruksha Guna (dry property) and Pitta vitiated due to Ushna Guna (hot property) and Tikshna Guna (penetrating or pungent property) are mainly responsible for reduction in cervical mucus. Treatment of Vandhyatva (infertility) is broadly classified into two groups i.e. Taila (oil) treated conditions or Ghrita treated conditions. The choice of Taila (oil) or Ghrita depends on the accompanying Doshas with Vata. If Vata is associated with Kapha, Taila (Acharya J.T., 1994) has to be preferred, while

in case of Pitta association, Ghrita (Acharya J.T., 2011) has to be preferred. Considering this Phalaghrita (Tripathi Brahmananda, 2010) was selected for the present trial Further, Basti is considered as sovereign remedy. Uttarbasti is one type of Basti which is all time best for the diseases of female. It tones up reproductive organs and improves the quality of Ambu i.e. cervical mucus. Thus by applying proper drug through Uttarbasti, disorders of female reproductive tract can be cured. Hence, this study was planned to evaluate the efficacy of Phalaghrita when given orally and by Uttarbasti on cervical factor responsible for infertility. MATERIALS AND METHODS: Selection of cases: Patients attending the O.P.D. of Prasuti – Streeroga (Gynaec) Department, fulfilling the criteria for selection, were included in the study. Total 32 patients of Vandhyatva (infertility) were screened for present study. Out of them 19 patients had known other factors of infertility, hence in the end they were excluded from the study and remaining 13 patients fulfilling the criteria were included randomly by simple random method in two groups among which 7 patients were registered in Group A and 6 patients were registered in Group B. All patients had completed the course of treatment. It was open labeled randomized interventional trial with efficacy. Ethical clearance The study was cleared by the Institutional Ethics Committee. Prior to initiation of the study, written consent was taken from each patient. Patients were asked to withdraw their name from the study at any time without giving any reason if they wish. Criteria for selection of cases: Patients of childbearing age having primary and secondary Infertility, having poor Moghissi mucus scale and abnormal Post coital test were registered for the study. Patients having infertility due to other than cervical factor, any

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urogenital infection, suffering from any chronic debilitating disease and sexually transmitted diseases were excluded from the study. Investigations: Hematological investigation:TLC, DLC, ESR

Hb%,

Urine:- Routine examination

and

Microscopic

Stool:- Routine examination

and

Microscopic

Moghissi mucus scale (Cervical mucus test) & Post Coital Test (PCT):- for diagnosis of Cervical Factor Endometrial biopsy, Hystero salpingo graphy and Trans Vaginal Grouping Drug A B

Dose

Route

Phalaghrita 15 gm/b. d. Oral Phalaghrita 5 ml for each Intra intra cervical cervical Uttarbasti

The follow up study of patients was conducted for one month after completion of the treatment. Criteria for Assessment The criteria for assessment of treatment were based on Sim’s Hunter and Moghissi’s score for cervical mucus and Post coital test (Mary G, 1992; Vaclav Insler Bruno Lunenfeld, 1993). 1. Amount: 0 = None 1 = 0.1 ml 2 = 0.2 ml 3 = ≥ 0.3 ml

Sonography:patients

for the exclusion of

Selection of the drug Phalaghrita is considered to be one of the best remedy for Infertility and it also has Deepana (appetizer), Pachana (digestive), Vatanulomana (putting in right direction i.e. downward direction of vayu ), Vrishya (aphrodisiac), Rasayana (rejuvenation), Balya (strength promoting) and Brimhana (Bulk promoting)properties thus Phalaghrita was selected for clinical trial. In this study, for oral group the Madhyam Matra (medium dose) i.e. 15gm. was selected of Phalaghrita and for Uttarbasti, only 5 ml of Phalaghrita was selected as uterine cavity has volume of only 3–4 ml.

Duration

Anupana

2 months Warm milk 6 days with 3 days interval after cessation _ of menses subsequently for two cycles 2. Spinnbarkeit: 0 = None 1 = 1-4 cm 2 = 5-8 cm 3 = ≥ 9 cm 3. Viscosity: 0 = Thick and highly viscous 1 = Intermediate type 2 = Mildly viscous 3 = Thin 4. Ferning: 0 = No crystallization 1 = Atypical 2 = Primary/Secondary 3 = Tertiary

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2 = 5 to 10 sperm/hpf 3 = 10 sperm/hpf

5. Cellularity: 0 = ≥11 cells/hpf 1 = 6 to 10 cells/hpf 2 = 1 to 5 cells/hpf 3 = No cells/hpf 6. Density of sperm: 0 = Dead sperm/No sperm 1 = 2 to 5 sperm/hpf

7. Motility of sperm: 0 = Immotile 1 = Insitu motile 2 = Sluggishly motile 3 = Rapid motile

Criteria for assessment of overall effect of therapy Total effect of therapy was assessed on the basis of percentage relief obtained. Cured

100% result, increase in cervical mucus score > 10 & conception

Markedly improved

75% result, increase in cervical mucus score > 10

Moderately improved Improved

50% result, increase in cervical mucus score > 5

Unchanged

0% result, no change increase in cervical mucus score after treatment

25% result, increase in cervical mucus score

Observation Total 13 patients were registered, among which 7 patients were registered in Group A and 6 patients were registered in Group B. All patients had completed the course of treatment. In this study, 84. 61% of patients had primary infertility and rest of patients had secondary infertility. Nearly, 30.76% belonged to age group of 23–27 years and 33–37 years respectively; 30.76% patients had 4–6 years chronicity. Maximum number of patients (92.30%) had regular menstrual cycle. Nearly 69.23% patients had moderate quantity of menses and rest of patients had history of scanty menses. 30.76% of the patients had associated complaint i.e. dysmenorrhoea. 53.84% patients had 4–5days duration of menstrual period. 76.92% had an interval of

26–30 days while 16.66 had an interval of 21– 25 days and 30–35 days respectively. RESULTS Status of 13 Patients of Vandhyatva (Cervical Factor) The effect of Phalaghrita orally in Group A showed that maximum relief was achieved on cervical mucus i.e. cellularity (73%), amount (62%), spinnbarkeit (56%), viscosity (52%), and ferning (30%) - Table 1, while on in density of sperm (86%), motility of sperm (82%)-Table 2. The effect of Phalaghrita Uttarbasti in Group B showed that maximum relief was achieved in amount (68%), cellularity (50%), spinnbarkeit (46%), viscosity (53%) and ferning (18%) –Table 3, while on density of sperm (66%), motility of sperm (59%)-Table 4.

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TABLE NO.1: Effect of Therapy on Factor of Cervical Mucus in Group A Sr. Factor of No. cervical mucus Amount 1 Viscosity 2 Ferning 3 Spinnabarkeit 4 Cellularity 5

Mean sore B.T.

A.T.

0.86 01 2 1 0.57

2.29 2.71 2.86 2.29 2.14

Relief (%)

S.D.

S.E.

‘t’

62 52 30 56 73

0.53 1.27 1.38 0.49 0.79

0.20 0.48 0.40 0.80 0.30

7.07 2.97 6 6.37 5.28

‘P’

0.001 0.05 0.001 0.001 0.01

TABLE NO.2: Effect of Therapy on Sperm Density and Motility in Group A Sr. Factor No. cervical mucus Sperm 1 Density Sperm 2 Motility

of Mean sore

Relief (%)

S.D.

S.E.

‘t’

‘P’

B.T.

A.T.

0.14

0.90

0.34

2.52

0.05

0.14

0.86

0.35

0.36

1.99

> 0.05

TABLE NO.3: Effect of Therapy on Factor of Cervical Mucus in Group B Sr. Factor of No. cervical mucus Amount 1 Viscosity 2 Ferning 3 Spinnabarkeit 4 Cellularity 5

Mean sore B.T.

A.T.

0.83 1.33 2.17 1.5 1.17

2.67 2.83 2.67 2.83 2.33

Relief (%)

S.D.

S.E.

‘t’

‘P’

68 53 18 46 50

0.41 0.84 0.55 0.82 0.98

0.17 0.17 0.22 0.33 0.40

11 4.93 2.24 4 2.31

0.001 0.01 > 0.05 0.01 > 0.05

TABLE NO.4: Effect of Therapy on Sperm Density and Motility in Group B Sr. Factor No. cervical mucus Sperm 1 Density Sperm 2 Motility

of Mean sore

Relief (%)

S.D.

S.E.

‘t’

‘P’

B.T.

A.T.

0.14

0.90

0.34

2.52

0.05

0.67

1.67

0.63

0.26

3.87

0.01

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Figure 1: Overall effect of therapy

50% 50.00%

42.85%

40.00%

33.33%

28.57% 30.00%

Group A

16.66% 20.00%

14.28%

Group B

10.00% 0% 0% 0% 0.00% A

A – Cured B – Markedly improved C – Moderately improved D – Improved E – Unchanged

In Group A, 28.57% of patients were conceived (cured), 14.28% of patients were markedly improved, 42.85% of patients were moderately improved and 14.28% of patients were improved. In Group B, 16.66% of patients were conceived (cured), 33.33% of patients were markedly improved and 50% of patients were moderately improved. None of the patients remained unchanged out of the 13 patients (figure 1). DISCUSSION Cervical factor is an important subset in infertility among women though it accounts only 5% of female infertility. According to Ayurveda, Ruksha Guna (dry property) of Vata and Ushna (hot) and Tikshna Guna of Pitta are two main factors of Cervical factor (scanty cervical mucus) and principles of the management of cervical factor are Bhrimhana (bulk promoting), Agnivardhaka (improving metabolism) and Vatanulamana (putting in right direction i.e. downward direction of vayu) treatment.

In the present study, maximum number of patients (30.76%) belonged to age group of 23– 27 years and 33–37 years respectively which is the most fertile period of women. The trend of late marriage is also increasing now a day because of higher education and social awareness of women. So, this may be probable causative factor and it is also because patients with infertility prefer Ayurvedic line of treatment after using modern medicine for a considerable duration. Nearly 30.76% patients had 4–6 years chronicity, it shows that, patients approach modern science and after failure or huge expenses they prefer to Ayurveda as their last hope, which will lead to chronicity of the disease. Statistically significant result were found on spinnbarkeit (p< 0.001), density of sperm (p<0.05), cellularity (p 0.01) and ferning of cervical mucus (p 0.001) which was better in group A than in Group B. statistically significant (p 0.001, 68%) result was found on amount of cervical mucus and motility of sperm (p<0.01) in Group B which was better than group A (Table 1, 2, 3 & 4).

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As per the result, it can be said that increase in amount of cervical mucus, spinnbarkeit, Motility and Density of sperms in Cervical mucus and Ferning in Group A, may be due to phytoestrogenic effect of Shatavari (Bopana N, 2007) and estrogenic property of Yastimadhu (Taro Nomura, 2002), Mishreya (Sharma PC, 2005; Albert-Puleo, M., 1980) present in Phalaghrita. Decrease in cellularity was observed in both the groups which may be due to anabolic effect of Shatavari (Chopra. R. N1986), Aswagandha (Chopra. R. N, 1986), Goghrita (Hariharannatha), etc. Wound healing effect of Haridra (T. K. Biswas, 2003) and Goghrita might have decreased the degenerative process of cervical mucus. Increment of amount of cervical mucus was observed in Group B due to its local regenerative effect on secretory unit of the cervix. The anabolic effect of Phalaghrita was observed on patients of infertility which was reported with increment of cervical mucus along with enhanced Agni. This is supported from the data that most of the patients taking Phalaghrita orally, also reported to have an increased appetite during treatment and also in the follow up period. It was also observed that the patients suffering from scanty menses before the treatment, they got relief particularly in Oral Group (Group A). It may be due to Vatapitta Shamaka property of drugs and phytoestrogenic

or estrogen property of Phalaghrita (Shatavari, Yastimadhu, Mishreya, etc.) after metabolism enters into blood, in hypothalamus estradiol is converted into catacholestrogen 2 hydroxylase enzymes. Catecholestrogen may influence GnRH release regulate hypothalamo pituitary ovarian axis and regulate Reproductive functions (Jeffcoat N, 2008; Bingham S., 1998; Brezinski A, 1996; Knight D,1996). Probable mode of action of Phalaghrita on cervical factors has been demonstrated in Figure 2. Phalaghrita orally is more effective than the Uttarbasti of Phalaghrita on the cervical factors. Phalaghrita corrects the Agni and pacify the vitiated Vata & Pitta when it is given orally. CONCLUSION Though occurrence of infertility due to cervical factor is 5% but it is very important causative factor due to its adverse effect on sperm penetration. The results of study reveal that oral administration of Phalaghrita is more effective than the Uttarbasti of Phalaghrita on the cervical factor. Also it can conclude that Phalaghrita has anabolic and regenerative action on whole of the genital tract and correct the Agni when it is given orally. Hence, Phalaghrita orally along with Uttarbasti is recommended for the patients of Infertility due to cervical factors.

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REFERENCES Acharya YT (2011), Charaka Samhita Sutra 13/15 with Ayurveda Deepika commentary by Chakrapanidutta, Chaukhambha Surbharati Prakashan Varanasi, reprinted edition, no.82.

Mary G. Hammond and Luther M.Talbert, (1992) Infertility – A practical guide for the physician, 3rd edition, published by Blackwell Scientific Publication, pg.324.

Albert-Puleo, M. (1980), Fennel and anise as estrogenic agents, J. Ethnopharmacol., Vol. 2 (4), PP. 337–344.

Sharma PC, Yelne MB, Dennis TJ (2005). Database of Medicinal Plants. Vol. 7. Ministry of Health and Family Welfare. Indian system of medicine and homiopathy. Govt. of India, New Delhi: Council of India, Ayurveda and Siddha :286.

Bingham S.(1998): Phytoestrogens, Where are we now? Br. 1. Nutr.79: 393–406. Bopana N, Saxena S. (2007) Asparagus racemosus – Ethnopharmacological evaluation and conservation needs. J Ethnopharmacol; 110:1–15. Brezinski, A Debi A. (1996) A phytoestrogens: The Natural” selectiveestrogen receptor modulators. Eur.1.Ob. andGyn. And Rep. Bio.85, 57–61. Chopra. R. N., Nayar. S. L. and Chopra. I. C. (1986), Glossary of Indian Medicinal Plants (Including the Supplement). Council of Scientific and Industrial Research, New Delhi.

Shastri Ambika dutta, (2007) Sushruta Samhita edited with Ayurveda Tattva Sandipika Hindi commentary , Part 1, 13th edition, reprinted, Chaukhambha Sanskrit Sanshana, Varanasi, Su.Sha.2/33. T. K. Biswas and B. Mukherjee,(2003) Plant medicines of Indian origin for wound healing activity: A review. Int J Low Extrem Wounds 2, 25–39. Taro

D. C. Dutta (2009), Textbook of Gynaecology including contraception, 5th edition revised reprint, published by New Central Book Agency, pg.223. Hariharannatha, Harita Samhita, (Ha. 1st Sthana 8/73) Jeffcoat N (2008): Sex Hormone therapy, In principle of Gynaecology(7th Edi.) Jaypee Brothers medical Publisher (P) LTD, New Delhi, pp.579–597. Knight D and Edan J.A.(1996): A Review of the Clinical Effect of Phytoestrogens. Obstet. Gynaecol. 87: 897. Source of Support: Nil

Nomura, Toshio Fukai, Toshiyuki Akiyama (2002). Chemistry of Phenolic Compounds of Licorice (Glycyrrhiza species) and theirestrogenic and cytotoxic activities. Pure Appl. Chem., Vol. 74, no. 7, p.1199–1206.

Tripathi Brahmananda (2010) Sharangdhara Samhita, Dipika Hindi commentary, Chaukhambha Surabharati Prakashana, Varanasi, reprinted edition, 9th chapter, pg.229–230. Vaclav

Insler Bruno Lunenfeld, (1993) Infertility male & female, 2nd edition, published by Churchil Livingstone, pg.60

Conflict of Interest: None Declared

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