Ethnobotanical Characteristics

Description

Perennial, climbing or prostrate; stem glabrous to pubescent. Leaf trifoliolate, petiole 6-20 mm long; leaflets 1.0-2.5 cm long and broad, rhomboid-ovate or rhomboid-obovate, mostly acute apiculate, pubescent above, pubescent on the veins and glandular punctate below, glands orange to black; stipules up to 3.5 mm long. Inflorescence a lax axillary raceme. Pedicel very short. Calyx 3.5-4.5 mm long, pubescent, teeth longer than the tube. Corolla yellow. Vexillum 5-6 mm long, pubescent externally. Fruit 1.2-2 cm long, 6-6.5 mm broad, glabrous, 2-seeded. (Jongbloed, 2003, e-Floras).

Habitat and Distribution

Habitat

Sand and gravel, Wadi. 

Distribution

Pakistan; India; Ceylon; Lower Burma; Equatorial Africa; West Indies; U.S.A; Australia. In UAE in the Northern Emirates. 

Part(s) Used

Whole plant

Traditional and Medicinal Uses

Used as abortive (Jongbloed, 2003).

Pharmacognosy and Phytochemistry

Plant material Studied

Dried leaf

General appearance

The leaf is obcordate in shape, grayish green with a short petiole and entire margins. It is small in size and has prominent venation. It has a smooth velvety touch and it is brittle but is usually found intact.

Microscopic characteristics

A transverse section through the leaf exhibits its isobilateral character, and palisade layers are found between the upper epidermis and above the lower epidermis. Both epidermises consist of small oval cells, and each epidermis is covered with numerous conical tapering trichomes, which are found more on the lower epidermis. Also, many of the trichomes are appressed to the epidermises. The upper epidermis is underlain by two layers of cylindrical compactly-arranged palisade cells that contain orange-brown or dark brown colored materials. The spongy mesophyll consists of many cells that have irregular shapes, and they are very rich in orange or orange-brown masses that may also separate from their cells. At the central part of the leaf, the spongy mesophyll cells surround grey vascular tissues with calcium oxalate prisms in the parenchyma cells, and their xylem vessels are annularly thickened. The lower palisade layer also contains orange-brown or dark brown colored materials.

Plant material Studied

Dried stem and branches

General appearance

The branches are cylindrical in outline, grey with a violet tint; the diameter about 2 mm. it is tomentose with a velvety touch.

Microscopic characteristics

A transverse section of the branch shows that it is circular in outline. It exhibits the following features starting from the epidermis inwards. The epidermal layer consists of compressed parenchyma cells that bear numerous conical tapering covering trichomes many of which are appressed to the epidermis; they also vary in length. A few short glandular trichomes with rounded heads are occasionally observed. The epidermis is underlain by 4-5 layers of yellow-colored compressed and distorted cortical parenchyma cells that surround a continuous circle of 3-4 layers of heavily lignified fibers that have thick walls and narrow lumens. These are underlain by a few layers of small, highly compressed cortical parenchyma cells that are light yellow. These parenchyma cells surround a continuous circle of heavily lignified vascular tissues that occupy a wide zone separated longitudinally by less lignified medullary rays. The vascular tissues contain wide pitted tracheids and annularly and reticulately thickened vessels. The pith consists of circular and polygonal cells that vary in size. These also enclose few orange-brown cells with thick walls.

Parts studied

stem and leaf

• A. TS through the stem showing the epidermal layer surrounding compressed and distorted cortical parenchyma, groups of heavily lignified fibers, a few layers of highly compressed parenchyma surrounding heavily lignified vascular tissues (dark in colour).
• B. TS of the leaf showing its isobilateral character, numerous conical tapering trichomes (more on the lower side), dark sponge mesophyll tissues with contents of orange or brown masses. 
• C. Leaf isolated covering trichomes (leaf) and isolated orange or dark brown masses found within the spongy mesophyll tissues (right). 

Chemical constituents

Steroidal glycoside (3-O-β-d-galactopyranosyl-stigmasta-5,22-diene) along with ergosterol peroxide, stigmasterol and lupeol. (Wasim Ahmed, 1992). Active chemical constituents, including alkaloids, glycosides, flavonoids, sterols and tannins (Mali 2008). Bergapten, isopimpinellin, umbelliferone and B-sitosterol from aerial parts. Proanthocyanidins from seeds. C6-glucosylapigenin (isovitexin), C6-glucosyl-C8-arabinosylapigenin, (schaftoside), di-C6, 8-glucosylapigenin (vicenin-2), and C6-glucisyl-C8-xylosylapigenin (vicenin-3) from leaves. lucenin -2 along with C-glycosides of vitexin, orientin, isoorientin and vicenin-2. (Rastogi, 1991, 1993, 1998).

The following chemical studies have been carried out on the plant Rhynchosia minima (Quality Control methods, 1998; Evans, 1996; ZCHRTM unpublished work):

Physicochemical constants 

Loss of weight in drying at 105°C :                                 8.10
Absolute alcohol solubility            :                                 4.40
Water solubility            :                                                 15.00

Successive extractives (%)

Petroleum ether (60-80) °C :                                         1.35
Chloroform :                                                                  2.10
Absolute alcohol :                                                         6.35

Ash values (%)

Total ash :                                                                     7.17
Water soluble ash :                                                       3.33
Acid insoluble ash (10% HCl) :                                     0.33

pH values (aqueous solution)

pH of 1% solution :                                                    6.583-6.588
pH of 10% solution :                                                   5.994-6.012

Elemental analyses

Ash values (British Herbal Pharmacopeia)
Assay and identification of element (AOAC International)

Apparatus		AA-6800 Shimadzu-Flame method
Element	Std. conc. mg/ml (ppm)	Sampleconc.mg/ml	Sample absorbance	Actual conc.mg/ml	Actual conc.( %)
Cr	1, 2, 4	10	0.0035	0.00313	0.000313
Zn	0.25, 0.5, 1	10	0.2329	0.04134	0.004134
Cu	1, 2, 4	10	0.0269	0.012	0.0012
Fe	1, 2, 4	10	0.5274	0.37355	0.037355
K	1, 2, 4	0.5	1.0192	36.886	3.6886
Pb	1, 2, 4	10	0.0000	0.0000	0.0000
Cd	0.25, 0.5, 1	10	0.0061	0.00122	0.000122
Ca	5, 10, 20	10	0.1145	3.67548	0.367548
1ppm conc. = 1µg/ml; Actual conc. (%) =Actual conc.(ppm)x0.0001 [1ppm=0.0001%]

UV Spectral studies

Ultraviolet Spectrum (USP reference)
Apparatus	Milton Roy Spectronic Genesys 5 Spectrophotometer - Milton Roy.
Sample conc. (mg / ml)	Solvent	λ max (nm)	λ min (nm)	Abs.( λ max - λ min)
1.25	Intestinal Fluid simulated without pancreatic pH=7.50.1	280 325	262 301	1.165 - 1.044 0.985 - 0.895
1	Gastric Fluid simulated without pepsin pH =1.20.1	274 331	256 319	0.878 - 0.767 0.578 - 0.568

Chromatographical Studies

Thin layer chromatography (TLC): Wagner and Bladt, 1996

 

 

TLC fingerprint of Petroleum ether 60-80°C (track 1) and MeOH extract (track 2)

Mobile phase Fig.	A&C	:	Toluene, ethyl acetate (93:7)
	B	:	Ethyl acetate, methanol, water (100:13.5:10)
	D	:	Toluene, ethyl formate, formic acid (5:4:1)
Detection	A	:	UV 366nm
Derivatization	B, C&D	:	Vanillin-Sulphuric acid-vis

Pharmacological and toxicological studies

The major constituents, along with antimicrobial activities against 12 bacterial and 4 fungal species, and the antioxidant activity of Rhynchosia minima essential oil were determined. The oil included isopropyl toluene, camphene and O-cymene. The essential oil exhibited marked activity against Escherichia coli, Staphylococcus aureus and Candida albicans, but was not active against Clostridium perfringens up to the concentration of100 μg/ml. The essential oil also exhibited anti-oxidant activity. The significant antimicrobial and antioxidant activities of R. minima oil suggests that it could serve as a source for compounds with therapeutic potential (Gundidza, 2009).
The petroleum ether and ethyl acetate extracts of Rhynchosia beddomei leaves showed inhibitory activity against some bacterial and fungal species at different concentrations (Bakshu, 2001)
The MeOH extract of the seeds of Rhynchosia volubilis (Leguminosae) showed antiproliferative activity against human gastric adenocarcinoma [MK-1, 50% growth inhibition (GI50): 25 microg/ml], human uterus carcinoma (HeLa, GI50: 30 microg/ml), and murine melanoma (B16F10, GI50: 8 microg/ml) cells. (Kinjo , 2001).
The seeds of Rhynchosia volubilis (SRV) (Leguminosae) and soybean have been used in oriental folk medicine to prevent postmenopausal osteoporosis. Their beneficial effects are attributed to a high content of isoflavone, which function as partial agonists or antagonists of estrogen (Kim, 2005).
The methanolic extracts of the flowers of Michelia champaca Linn. (Champaka), Ixorabrachiata Roxb (Rasna) and Rhynchosia cana Willd were possess significant anti-inflammatory activity against cotton pellet induced subacute inflammation in rats. (Dahanukar, 2000).
Rhynchosia sublobata (root), was screened for activity against Plasmodium falciparum and Human Immunodeficiency Virus Type 1 (HIV-1, IIIB strain) along with many other plants and a positive activity value was observed. (Maregesi,2010)
The following pharmacological and safety evaluation studies were carried out at ZCHRTM on the plant extract of Rhynchosia minima (Derelanko 2002; Han, 2003).

ACTIVITY	RESULTS
	Strong	Moderate	Mild	Negative
Anti-diabetic activity				√
Antidepressant activity (TST)	√
Anticoagulant effect		√
Anti-gastric ulcer activity		√
Effect on Guinea pig ileum		√
Effect on rabbit jejunum	√
Effect on right rat atria			√
Blood pressure and heart rate			√
Effect on rat fundus	√
Acute toxicity studies				√
Autonomous activities				√
Biochemical studies				√
Hematological studies (HGB & HCT) ↑	√
Vital organs’ weight				√
Locomotor activity test				√
Motor co-ordination (grip strength & motor activity				√
Rectal temperature				√
Body weight				√
Mortality				√

Summary of the results

Showed antidiarrheal/ anti-secretory activity; produced significant inhibition of gastric mucosal damage; Antidepressant activity; and elevated the hemoglobin and hematocrit. Toxicity assessment is safe at the dose tested; No mortality was recorded.

Antimicrobial activity

The aqueous extract of the whole plant was tested against Mycobacterium smegmatis, Candida tropicalis, different strains of Staphylococcus aureus (Including ATCC 257) as well as strains of Methicillin Resistant Staphylococcus aureus, strains of Streptococcus pyogenes, strains of E. coli (Including ATCC UN 109), different strains of ESBL-producing K. pneumonia, E. coli, Pseudomonas aeruginosa. A weak antibacterial activity against S. aureus, S. pyogenes, M. smegmatis was observed. 

References

• Bakshu, L M. Venkata Raju, R R Antimicrobial activity of Rhynchosia beddomei Fitoterapia. Jun 2001; 72: 579-82
• J.R.I.Wood. A handbook of the Yemen flora, Royal Botanic Garden, Kew, p.161, 1997.
• Jonbloed, M. V., Feulner, G. R., Boer, B. & Western, A. R. (2003). The comprehensive Guide to the Wild Flowers of the United Arab Emirates, Erwda, Abu Dhabi, U.A.E. 
• Kim J, Um SJ, Woo J, Kim JY, Kim HA, Jang KH, Kang SA, Lim BO, Kang I, Choue RW, Cho Y. Comparative effect of seeds of Rhynchosia volubilis and soybean on MG-63 human osteoblastic cell proliferation and
• estrogenicity. Life Sci. Epub, Aug 2005, 78:30-40.
• Kinjo J, Nagao S, Tanaka T, Nonaka GI, Okabe H. Antiproliferative constituents in the plant 8. Seeds of Rhynchosia volubilis. Biol Pharm Bull. Dec 2001;24(12):1443-5.
• M. Gundidza, N. Gweru, M. L. Magwa, N. J. Ramalivhana, G. Humphrey, A. Samie and Mmbengwa V Phytochemical composition and biological activities of essential oil of Rhynchosia minima (L) (DC) (Fabaceae)
• African Journal of Biotechnology Vol. 8 (5), March 2009, 8: 721–724, 6. 
• Mali RG and Mahale NB. Evaluation of Rhynchosia minima (Linn.) DC Leaves for Anthelmintic Activity. International Journal of Pharmaceutical Sciences and Nanotechnology Volume1, Issue 2 ,July - September 2008.
• Maregesi S.,Van Miert S.,Pannecouque C.,Feiz Haddad M.H.,Hermans N.,Wright C.W.,Vlietinck A.J.,Apers S.,Pieters L. Screening of tanzanian medicinal plants against plasmodium falciparum and human
• immunodeficiency virus Planta Medica, vol./is. 2010, 76/2(195-201), 0032-0943;1439-0221 
• Rastogi & Mehrotra, Compendium of Indian medicinal plants: vol. 2, PID, New Delhi,1991,p.587.
• Rastogi & Mehrotra, Compendium of Indian medicinal plants: vol. 5, PID, New Delhi,1998, p.725
• Rastogi & Mehrotra, Compendium of Indian medicinal plants: vol. 4, PID, New Delhi, 1993, p.634.
• S.A. Dahanukar, R.A. Kulkarni, N.N. Rege. pharmacology of medicinal plants and natural products Indian Journal of Pharmacology 2000; 32: 81-118.
• Wasim Ahmed, Zaheer Ahmad and Abdul Malik. Stigmasteryl galactoside from Rhynchosia minima. Phytochemistry, Volume 31, Issue 11, 1992, Pages 4038-4039
• Western, A. R. (1986).  The Flora of United Arab Emirates. An introduction. –Al Ain.
• Western, A. R. (1989). The Flora of United Arab Emirates. An introduction. Publications of the U.A.E. University.