Abstract
Objective:
To establish the pharmacognostic standards for the correct identification and standardization of an important Antidiabetic plant described in Ayurveda.
Materials and Methods:
Standardization was carried out on the leaf and stem of Basella alba L. with the help of the macro-morphological, microscopic, physicochemical and qualitative phytochemical studies.
Results:
Several specific characters were identified viz. clustered calcium oxalate crystals in the cortex region, absence of trichomes, succulent, thick, mucilaginous, fibrous stem. Rubiaceous type of stomata on both sides of the leaf. Quantitative microscopy along with physicochemical and qualitative phytochemical analysis were also established.
Conclusion:
The pharmacognostic standards could serve as the reference for the proper identification of the Basella alba L. which is an important anti-diabetic plant described in Ayurveda.
Keywords: Anti-diabetic, Basella alba, microscopy, standardization, thin layer chromatography
Introduction
Upodikā commonly known as Bāyibasale soppu in Kannada (local language, Karnataka, India) which is botanically equated to Basella alba L. belonging to family Basellaceae, which is an extremely heat tolerant fast growing perennial vine.[1,2,3] It is also known as Malabar spinach, Indian spinach, Ceylon spinach, country spinach and wine spinach. It is a glabrous herb, fleshy, perennial, much branched creeping climbing herb. Leaves simple, alternate, petiolate, sub succulent, broadly ovate, acute or acuminate, cordate at base. Inflorescence in spike, flowers white or red, bracteoles longer than perianth. Fruits red, white, black, globose, utricle enclosed in the perianth [Figure 6]. It is cultivated at homes as a pot herb because of its high nutritive value.[4] It is further observed that two common species i.e., the red-stemmed B. rubra and the green-stemmed B. alba is found in India which are indistinguishable in taste. Ayurveda recommends Basella in the treatment of several conditions such as anaemia, cough, dysentery and diarrhoea and as a poultice and in the treatment of mouth ulcers in south India.[5]
Figure 6.
Basella alba - Habitat
B. alba is reported to contain several phytoconstituents such as proteins, alkaloids, carbohydrates, polysaccharides, phenols, flavonoids, carotenoids, minerals and vitamins.[4,6,7] It is also an important ethno veterinary medicinal plant used for the treatment of retained after birth and anaplasmosis and administered in balanitis and gonorrhoea. The mucilaginous liquid obtained from the leaves and tender stalks of this plant is a remedy for habitual headaches. A decoction of the leaves is a good laxative for pregnant women and children,[8] apart from this, it was also reported that daily consumption of B. alba has a positive effect on total-body vitamin A stores in men.[9] It has immense potential activity on diabetes and this has been scientifically well established. The aqueous extract of B. alba leaf exhibited anti-diabetic effect on alloxan induced diabetic rats.[10] The mucilage present in B. alba has been credited with hypoglycaemic activity.[11] It also possess CNS depressant activity, gastro protective activity,[12,13] anti-inflammatory activity,[14,15] membrane stabilization property in human blood cell membrane,[16] antibacterial activity,[17] wound healing and anti-ulcer activity.[18,19] B. alba is consumed in food preparations in southern India. In Ayurveda the drug is used for various indications either alone or in combination for mada (intoxication), anidrā (insomnia), paādadari (cracked feet), jvara (fever), pravaāhikaā (dysentery), arśhas (haemorrhoids), śiātapitta (urticaria), vranaśotha (inflammatory), arbuda (tumours), raktapitta (bleeding disorders).[20,21,22] The plant is used in Ayurveda treatment for cancer management.[23] Pharmacognostic profile of the B. alba hasn‘t yet been carried out and hence the present work focuses on its pharmacognostic profile.[24]
Materials and Methods
Plant collection and processing
The fresh samples of plants Upodikā (Basale soppu in Kannada language, Karnataka, India)-Basella alba L., (leaf and stem), were collected from different vegetable markets of Bangalore (K. R. Puram, Krishna Rajendra and Jayanagar vegetable markets, Bangalore, India) and the plants were identified with the help of Bangalore Flora and from Survey of Medicinal Plant Unit, NADRI, Bangalore. The plant material was processed in the form of herbarium, and deposited in RRCBI Herbarium, Bangalore for the future reference (Voucher Specimen 12357). The plant material was washed with water removal of foreign objects and shade dried. It was then milled to coarse powder, kept in paper bags and stored at ambient temperature until use.
Organoleptic evaluation
Organoleptic evaluation was done by observing the leaf and stem with the naked eyes and taking note of the colour, size, odour and other diagnostic parameters.
Microscopic, histochemical and histological analysis
Different parts of the plants such as stem and leaf were soaked in 70% alcohol for 24 hours, and free hand sections were taken, cleared with chloral hydrate solution and water, stained with saffranin according to the standard prescribed methods. Photomicrographs were captured with Nikon Digital camera and Cat cam camera. Powder and Maceration studies were also carried out following the standard methods.[25,26]
Quantitative microscopy
Quantitative studies were carried out for stomatal index, vein islet number and palisade ratio from laminar region and then mean average values were calculated from different observations, followed by standard methods.[27]
Physico-chemical analysis
Physico-chemical analysis such as ash values, extractive values were carried out according to the standard procedures prescribed in Ayurvedic Pharmacopeia of India and preliminary phytochemical screening was carried out as per the standard methods and procedure.[27]
Phytochemical screening
Preliminary phytochemical screening was carried out for different extracts by using standard procedures.[28]
Thin layer chromatography profile
Dried stem and leaf samples were extracted with petroleum ether, chloroform and methanol at room temperature with rotary shaker. TLC studies of these extracts were carried out by using commercially available precoated plates with standardized adsorption layers, i.e., Silica gel 60 F254, (Merck, Germany). All the solvents systems were selected by trial and error basis. The chromatograms were developed in twin through glass chambers on 10 × 10 cm plates till the mobile phase travelled up to a distance of 8 cm from starting point. After development, the plates were dried at room temperature for 5-10 minutes and observed under UV-254. Photographs were taken and the Rf values were recorded.[29]
Results
Macroscopic characters
Dried stem was pale green, compressed. Mature stem was hard, fibrous, hard to break up by hand, surface rough, winged, and triangular. Petiole 1-3 cm long, midrib protruding at the lower side and grooved at the upper side, odour pleasant and taste slightly sweetish. Leaves were simple, thick, 3-9 × 2-8 cm, smooth above, glaucous beneath, base was shallowly cordate or round, margin entire, apex acuminate, smell agreeable, tasting sweetish and mucilaginous.
Microscopic characters of stem, petiole and leaf
T.S. of stem shows star shaped with five extended wings with single layer of epidermis, made up of parenchymatous cells covered by a thin layer of cuticle, followed by an epidermis of two to three layers. The hypodermis is composed of compactly arranged polygonal parenchymatous cells without any inter cellular spaces. Besides the hypodermis, cortex region is formed by 6-10 layers of compactly arranged polygonal thin walled parenchymatous cells followed by the cortex, 3-4 layers of compactly arranged sclerenchymatous layer of cells (pericycle), where vascular bundles are collateral, conjoint and closed. In the centre portion, pith region shows compactly arranged parenchymatous cells with simple and compound starch grains in groups and with clustered calcium oxalate crystals [Figure 1].
Figure 1.
(a) T.S. of stem × 10 × ×4, (b) enlarged portion showing prominent pericycle and vascular bundle × 10 × ×10. cu: cuticle, epi: epidermis, pi: pith, scl: sclerenchyma, vb: vascular bundle
T.S. of petiole is strongly convex and spherical on the lower side. A central narrow groove on the upper surface and two lateral narrow winged extensions on either side and shows a single layer of epidermis. Epidermis made up of single layer of rectangular cells covered with thin cuticle. It is followed by 8 to 10 layers of tangentially elongated thin walled parenchymatous cells forming the ground tissue. Calcium oxalate crystals abundantly present in this region. Most of the calcium oxalate crystals present in the narrow winged extensions and some crystals are present close to the vascular bundle region. The collateral meristele shows 3-7 vascular bundles that face each other laterally [Figure 2a and b].
Figure 2.
(a) T.S. of the petiole, (b) T.S. of the petiole portion enlarged, (c) T.S. of the leaf through midrib region, (d) T.S. of the leaf through laminar region. avb: accessory vascular bundle, cor: cortex, cu: cuticle, cr: crystals, epi: epidermis, lepi: lower epidermis, pa: parenchyma, pal: palisade, ph: phloem, spar: spongy parenchyma, uepi: upper epidermis, vb: vascular bundle, xy: xylem
T.S. of the leaf through midrib region [Figure 2c] was convex at the lower side and grooved at the upper side, young leaf showed bicollateral meristeles in the centre, getting connected forming a single meristele encircled by a parenchymatous ring of pericycle in mature leaf. Parenchyma cells are 3-4 layers are in the narrow groove at the upper side, 5 to 6 layers towards the lower side. These cells are polygonal and compactly arranged without any air spaces. Clustered calcium oxalate crystals are present abundantly in the ground tissue region of the leaf. Most of the calcium oxalate crystals are present in the narrow winged extensions in laminar region, few crystals are also present close to meristele region. The single layered epidermis cells are rectangular, arranged compactly and covered by a thin cuticle and traversed with stomata. The laminar mesophyll region showed one to two layers of compactly arranged palisade parenchyma cells, two to four layers of loosely arranged spongy parenchyma cells, the calcium oxalate crystals were present abundantly in the upper layer of palisade parenchyma cells and were absent in the spongy parenchyma cells. Spongy parenchyma cells showed simple and compound starch grains in groups [Figure 2d].
Powder microscopy of stem, leaf and maceration study
Stem powder was light green in colour, smell agreeable, taste slightly sweetish and mucilaginous. Under microscope, it exhibits fragments of different tissues. Epidermal cells in surface view which were polygonal and tangentially elongated thin walled parenchymatous cells, single rosette calcium oxalate crystals, helical vessels, pitted xylem vessels, spiral xylem vessels, reticulate and thickened helical vessels in groups, elongated fibre and parenchymatous cells, single stomata and prism shaped crystals [Figure 3].
Figure 3.
Powder microscopy of stem: (a) Powder macroscopy, (b) epidermal cells in surface view, (c) calcium oxalate crystals, (d) spiral and helical vessel, (e) pitted xylem vessel, (f) spiral xylem vessel, (g) reticulate xylem vessel, (h) Elongated fiber and parenchymatous cells
Leaf powder was light green, rough to touch when treated with chloral hydrate solution. When observed under the microscope, it showed different fragments of tissues. Powder showed different fragments of tissues such as upper and lower epidermal cells in surface view, prism shaped crystals, reticulate xylem vessels, clustered calcium oxalate crystals, helical xylem vessel, abundant starch grains, epidermal cells with stomata, parenchyma cells and spiral and pitted lignified xylem vessels [Figure 4].
Figure 4.
Powder microscopy of leaf: (a) Leaf powder macroscopy, (b) epidermal cells in surface view, (c) prism crystals, (d) reticulate vessel, (e) crystals, (f) helical xylem vessel, (g) starch grains, (h) epidermal cells with stomata
Quantitative microscopy
Quantitative studies were carried out for stomatal ratio, stomatal index, vein islet number and palisade ratio from the laminar region and then the mean average values were calculated from different observations. The results are given in Table 1. Measurements of different tissues of leaf and stem were carried out, and the results are presented in the Tables 2 and 3.
Table 1.
Quantitative study of Basella alba leaf
Table 2.
Micrometric details of Basella alba stem
Table 3.
Micrometric details of Basella alba leaf
Macerate of stem shows disintegrated tissues of polygonal epidermal cells with stomata in the surface view, fibers with pits, groups of fibers, abundant and clustered calcium oxalate crystals, parenchyma cells in groups and single, reticulate vessel, lignified helical and spiral xylem vessels. Macerate of the leaf shows disintegrated different tissues such as epidermal cells, stomata in the surface view, parenchyma cells in groups, abundant clustered calcium oxalate crystals, helical and spiral xylem vessels.
Physico-chemical analysis
Total ash and acid insoluble ash values give an idea about the earthy/silica matter present in the stem and leaves of the drug. The alcohol and water extractive values obtained were initially useful for knowing the quality of the drug. The 5% aqueous pH values showed it to be neutral, i.e., the drug is consumable. The results obtained were recorded in Tables 4 and 5.
Table 4.
Physicochemical parameters
Table 5.
Extractive values by Soxhlet extraction
Preliminary phytochemical analysis
Preliminary phytochemical screening revealed the presence of alkaloids, carbohydrates, phenols, proteins, tannins, starch etc., and the results obtained were recorded in Table 6.
Table 6.
Preliminary phytochemical tests for different extracts of Basella alba leaf
Thin layer chromatography
TLC studies were performed for the development of characteristic finger print profile for different extracts. The powdered drug (2 g in 25 ml) was extracted with different solvents such as petroleum ether, chloroform and methanol at room temperature. These extracts were used to establish the TLC fingerprints by using different solvent systems on trial and error basis such as Toluene: Hexane: Ethyl acetate - 6:3.5:0.5 for petroleum ether extracts [Figure 5a–d], Toluene: Ethyl acetate: Chloroform - 6:3.5:0.5 for chloroform extracts [Figure 5e–h] and Ethyl acetate: Methanol: Water - 7.7:1.5:0.8 for methanol extracts of both stem and leaves [Figure 5i–l]. After developing, the plates were dried under room temperature for 5-10 minutes and observed under UV-254 and sprayed with Anisaldehyde-H2SO4 reagent. Photographs were taken and the Rf values are recorded in Table 7. The Rf values obtained are invariable for the studied extracts with the solvent systems used, hence any change in these is an indicative of adulteration of the drug. These TLC finger prints may be used as standard parameter during the standardization of the drug upodikā.
Figure 5.
TLC fingerprint studies: (a-d) Petroleum ether extract of stem and leaves under UV-254 and after derivatization, (e-h) chloroform extract of stem and leaves under UV-254 and after derivatization, (i-l) Methanol extract of stem and leaves under UV-254 and after derivatization
Table 7.
Rf Values of Stem and Leaf
Discussion
Pharmacognostic studies on the leaves and stem of B. alba which has got a very potent anti-diabetic property have been carried out. Plant is rich in mucilage, vitamin C with antioxidant properties. Morphology as well as various pharmacognostic aspects of the leaf as well as stem were studied along with phytochemical and TLC studies. All these parameters are useful in authentication of a genuine drug for the detection and prevention of adulteration. Leaf and stem of B. alba exhibits a set of diagnostic characters, which will help identify the drug in a fragmented form as well as in whole form. The main diagnostic features of the leaf and stem are summarized as: Presence of clustered calcium oxalate crystals in the cortex region, absence of trichomes, presence of succulent, thick, glabrous, mucilaginous, fibrous stem; conjoint, collateral, endarch and closed vascular bundles; 3-4 layers of sclerenchymatic pericycle; small, rounded, simple and compound starch grains in the pith and cortex region; helical pitted and reticulate xylem vessels in powdered tissues; abundant clustered calcium oxalate crystals in the leaf mesophyll region, and above the vascular bundles in the cortex region; succulent, mucilaginous leaf with cordate base without any trichomes; Rubiaceous (paracytic) type of stomata on both sides of the leaf; presence of clustered calcium oxalate crystals and simple, compound starch grains in the petiole as well as in the leaf; presence of collateral conjoint and closed vascular bundle in the leaf as well as in the petiole; one to two large median bundles in the mid rib region enclosed by 2-4 layers of collenchymatous tissues; reticulate and spiral xylem vessels in powder and macerate tissues.
B. alba has got different medicinal uses apart from the anti-diabetic properties. Further safety and efficacy studies along with and advance phytochemical studies are required, for further validation of the drug. We have highlighted the important diagnostic characters through macro and microscopic studies which will be useful for identification of valuable medicinal and nutritive traditional plant both in dry and fresh form. It is further observed that two common species i.e., the red-stemmed Basella rubra and the green-stemmed B. alba is found in India which are indistinguishable in taste and colour. Other botanical sources like Portulaca oleracea L. and Talinum fruiticosum L. (Juss) (= T. triangulare) are also known as Pasalikiārai in Tamil. The present study has listed many parameters based upon which B. alba can be identified.
Financial support and sponsorship
Intra Mural Research Project of CCRAS, Ministry of AYUSH, Government of India.
Conflicts of interest
There are no conflicts of interest.
Acknowledgements
We are highly thankful to Central Council for Research in Ayurvedic Sciences (CCRAS), Ministry of AYUSH, New Delhi, for the sanction of the project and for providing the facilities to carry out the work successfully.
References
- 1.Grubben GJ, Denton OA. Plant resources of tropical Africa. Vegetable. Vol. 4. Wageningen, Leiden: PROTA Foundation, Backhuys, CTA; 2004. pp. 103–11. [Google Scholar]
- 2.Rathee S, Ahuja D, Rathee P, Thanki M, Rathee D. Cytotoxic and antibacterial activity of Basella alba whole plant: A relatively unexplored plant. Pharmacol Online. 2010;3:651–8. [Google Scholar]
- 3.Deshmukh SA, Gaikwad DK. A review of the taxonomy, ethnobotany, phytochemistry and pharmacology of Basella alba (Basellaceae) J Appl Pharm Sci. 2014;4:153–65. [Google Scholar]
- 4.Anonymous. The Wealth of India, Raw Materials. Revised Edition. Vol. 2. New Delhi: Publications and Information Directorate, Council of Scientific and Industrial Research; 1988. pp. 50–2. [Google Scholar]
- 5.Baskaran T. It Isn't Spinach, but it's as Nutritious. The Hindu. [Last accessed on 2012 Aug 08]. Available from: http://www.thehindu.com/features/metroplus/Food/it-isnt-spinach-but-its-asnutritious/article3742102.ece .
- 6.Anonymous. Reviews on Indian Medicinal Plants. Vol. 4. New Delhi: Indian Council of Medical Research; 2004. pp. 96–101. [Google Scholar]
- 7.Kumar S, Prasad AK, Iyer SV, Vaidya SK. Systematic pharmacognostical, phytochemical and pharmacological review on an ethno medicinal plant, Basella alba L. J Pharmacogn Phytother. 2013;5:53–8. [Google Scholar]
- 8.Daniyan SY, Muhammad HB. Evaluation of the antimicrobial activities and phytochemical properties of extracts of Tamaridus indica against some diseases causing bacteria. Afr J Biotechnol. 2008;7:2451–3. [Google Scholar]
- 9.Haskell MJ, Jamil KM, Hassan F, Peerson JM, Hassain MI, Fuchs GJ, et al. Daily consumption of Indian spinach (B. alba) or sweet potatoes has positive effect on total-body Vitamin A store in Bangladeshi men. Am J Clin Nutr. 2004;80:705–14. doi: 10.1093/ajcn/80.3.705. [DOI] [PubMed] [Google Scholar]
- 10.Bamidele O, Akinnuga AM, Olorunfemi JO, Odetolo OA, Oparaji CK, Ezelgbo N. Effects of aqueous extract of Basella alba leaves on haematological and biochemical parameters in albino rats. Afr J Biotechnol. 2010;9:6952–5. [Google Scholar]
- 11.Palanuvej C, Hokputsa S, Tunsaringkarn T, Ruangrungsi N. In vitro glucose entrapment and alpha glucosidase inhibition of mucilaginous substances from selected Thai medicinal plants. Sci Pharm. 2009;77:837–49. [Google Scholar]
- 12.Anandarajagopal A, Sudhakar D, Ajaykumar TV, Muthukumaran G. Evaluation of CNS depressant activity of aerial parts of Basella alba L. IJPIS J Pharmacol Toxicol. 2011;1:1–6. [Google Scholar]
- 13.Kumar V, Bhat ZA, Kumar D, Khan NA, Chashoo IA, Ara I. Gastroprotective effect of leaf extracts of Basella alba var albaagainst experimental gastric ulcers in rats. Braz J Pharmacogn. 2012;22:657–62. [Google Scholar]
- 14.Krishna CB. Anti inflammatory activity of Basella alba L. in albino rats. J Appl Pharm Sci. 2012;2:87–9. [Google Scholar]
- 15.Rodda R, Kota A, Sindhuri T, Kumar SA, Gnananath K. Investigation on anti inflammatory property of Basella alba L. leaf extract. Int J Pharm Pharm Sci. 2012;4:452–4. [Google Scholar]
- 16.Kumar V, Bhat ZA, Kumar D, Bohra P, Sheela S. In vitro anti inflammatory activity of leaf extracts of Basella alba Linn. var. Int J Drug Dev Res. 2011;3:176–9. [Google Scholar]
- 17.Azad AK, Wan Azizi WS, Babar ZM, Labu ZK, Zabin S. An overview on phytochemical, anti-inflammatory and anti-bacterial activity of Basella alba leaves extract. J Sci Res. 2013;14:650–5. [Google Scholar]
- 18.Mohammed HK, Abraham A, Saraswathi R, Mohanta GP, Nayar C. Formulation and evaluation of herbal gel of Basella alba for wound healing activity. J Pharm Sci Res. 2012;4:1642–8. [Google Scholar]
- 19.Venkatalakshmi P, Senthamaraiselvi V. Anti ulcer effect of Basella alba leaf extract in aspirin induced albino rats. Int J Pharm Sci Res. 2012;3:2539–42. [Google Scholar]
- 20.Acharya YT, Agnivesha . Commentary by Chakrapanidatta. Charakasamhita of Charaka, Chikitsasthana. 14, Ver. 133-135. Varanasi: Chaukhamba Press; 2011. p. 507. Reprint. [Google Scholar]
- 21.Acharya YT, Agnivesha . Commentary by Chakrapanidatta. Charakasamhita of Charaka, Chikitsasthana. 19, Ver. 31-33. Varanasi: Chaukhamba Press; 2011. p. 551. Reprint. [Google Scholar]
- 22.Misra BS, editor. Purvardha (Part 1). Bhavaprakasha of Bhavamishra, Shakavarya-10. 9th ed. Varanasi: Chaukamba Sanskrit Samstana; 2005. pp. 665–836. [Google Scholar]
- 23.Shruti SD, Roshan A, Naveen KH. A review on medicinal importance of Basella alba L. Int J Pharm Sci Drug Res. 2012;4:110–4. [Google Scholar]
- 24.Adhikari R, Kumar N, Shruthi SD. A review on medicinal importance of Basella alba L. Int J Pharm Sci Drug Res. 2012;4:110–4. [Google Scholar]
- 25.Trease GE, Evans WC. Pharmacognosy. London: Bailliere Tindall; 1971. [Google Scholar]
- 26.Johansen DA. Plant Microtechnique. New York: McGraw Hill; 1940. [Google Scholar]
- 27.Anonymous. Quality Control Methods for Medicinal Plant Materials. Geneva: World Health Organization; 1998. [Google Scholar]
- 28.Anonymous. Physico-chemical Standards of Unani Formulations. Part-IV. New Delhi: Central Council for Research in Unani Medicine (CCRUM), Department of AYUSH, M/o Health and Family Welfare, Government of India; 2006. pp. 157–60. [Google Scholar]
- 29.Sethi PD. High Performance Thin Layer Chromatography (HPTLC) 1st ed. New Delhi: CBS Publishers and Distributors; 1996. pp. 1–74. [Google Scholar]