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 Table of Contents  
ORIGINAL ARTICLE
Year : 2011  |  Volume : 3  |  Issue : 2  |  Page : 249-252  

Trichosanthes dioica Roxb.: Pharmacognostic standardization of the female leaves with special emphasis on the microscopic technique


1 Department of Pharmacognosy, Lovely School of Applied Medical Sciences, Lovely Professional University, Jalandhar, Punjab, India
2 Institute of Post Graduate Teaching & Research in Ayurveda, Gujarat Ayurved University, Jamnagar, Gujarat, India
3 Department of Pharmacology, University Institute of Pharmaceutical Sciences, Punjab University, Chandigarh, India

Date of Submission23-Nov-2010
Date of Decision29-Dec-2010
Date of Acceptance05-Jan-2011
Date of Web Publication12-May-2011

Correspondence Address:
Lalit Machawal
Department of Pharmacology, University Institute of Pharmaceutical Sciences, Punjab University, Chandigarh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0975-7406.80780

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   Abstract 

Objectives: Standardization is essentially a measure for ensuring the quality control of herbal drugs. But the gender of the plant affecting the quality of crude drugs does not appear to have been taken care of so far. Today, sophisticated modern research tools for the evaluation of the plant drugs are available but the microscopic method is one of the simplest and cheapest methods. Methods and Materials: The pharmacognostic investigation of the fresh, powdered, and anatomical sections of the female leaves of Trichosanthes dioica Roxb. was carried out to determine its bioprospective constants. Results and Discussion : Externally, the leaves possess a cordate base, a sinuate and dentate margin, an acute to acuminate apex, and both surfaces are very rough with rigid hair surface. Internally, it shows the presence of anomocytic stomata, unicellular, and both glandular and simple covering trichomes scattered as such throughout or attached with the cells of the epidermis. Majority of the glandular trichomes are with a four-to-five-celled uniseriate stalk and unicellular head; very few are short and with uni- to bicellular stalk and uni- to multicellular head especially from that of the petiole region. Simple covering, multicellular, uniseriate thick-walled trichomes are of various sizes. Usually cells of both simple and glandular trichomes are often embedded with cystolith. Phytochemical studies of the powdered leaves revealed the presence of alkaloids, resins, glycosides, flavonoids and some carbohydrates. The pharmacognostic profile of the leaves will assist in the standardization for quality, purity, and sample identification.

Keywords: Female leaf morphology, pharmacognostic standardization, Trichosanthes dioica


How to cite this article:
Singh S, Chauhan M G, Machawal L. Trichosanthes dioica Roxb.: Pharmacognostic standardization of the female leaves with special emphasis on the microscopic technique. J Pharm Bioall Sci 2011;3:249-52

How to cite this URL:
Singh S, Chauhan M G, Machawal L. Trichosanthes dioica Roxb.: Pharmacognostic standardization of the female leaves with special emphasis on the microscopic technique. J Pharm Bioall Sci [serial online] 2011 [cited 2019 May 21];3:249-52. Available from: http://www.jpbsonline.org/text.asp?2011/3/2/249/80780

Trichosanthes dioica Roxb. (family Cucurbitaceae) is a dioecious climber with a perennial root stock. It is found wild in the plains of North India from Punjab to Assam. [1],[2],[3] A brief description of T. dioica is found in the Charaka Samhita, [4] Sushruta Samhita, [5] and Sangraha Samhita. [6] T. dioica had a blood sugar lowering effect in experimental rat models [7] and mild diabetic human subjects, [8] and also an antifungal [9] and antibacterial activity. [10] To maintain the quality and purity, we report the macro- and microscopic and some other pharmacognostic characters for the female leaves of the T. dioica, which could be used to prepare a monograph for the proper identification of the plant.


   Materials and Methods Top


Fresh female plants of T. dioica Roxb. were collected in the month of July 2005 from the cultivated field of Anand Agriculture University (Gujarat). Plant leaves' authenticity was carried out by Gujarat Ayurvedic University.

Morphology of the leaf

The detailed morphology of fresh leaves of female plants of T. dioica Roxb. was studied [Figure 1] and their characters are in given in [Table 1].
Figure 1: Detailed section of the female leaf

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Table 1: Macroscopic characters of female leaves

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Microscopic characters

Transverse section of female leaves

A diagrammatic T.S. passing through the midrib projects strongly at the lower side and elevated at the upper side; three to four meristeles of various sizes encircled by the sclerenchymatous band lie in the centre and the lamina is dorsiventral. The layer of the upper and lower epidermis is with a thick cuticle and stomata. The cells of the upper epidermis unlike the lower one are lignified in sections. It bears simple uniseriate and multicellular and glandular trichomes with a unicellular stalk and multicellular head and with multicellular stalk and unicellular head, embedded with yellowish contents. The simple trichomes are straight or bent or often resting on a pedestial base and are also embedded with cystolith; leaf trichomes are lignified. Collenchymatous tissue lies underneath both the upper and lower midrib region, it being one to two layered only; at the lateral sides of the lower midrib region, the bicollateral meristele of various sizes lies in the center, the lowest being bigger in size and is composed of radially arranged xylem vessels associated with tracheids, fibers, and medullary rays. Except the upper most meristele lying underneath the collenchymatous tissue, all other meristeles are encircled by a continuous band of sclerenchyma fibers. The lamina is composed of the upper and lower epidermis with cuticle and bear trichomes. Under the upper epidermis lies a layer of palisade cells; the remaining cells of the mesophyll consist of three to four rows of spongy parenchyma traversed with obliquely cut vascular bundle.

Transverse section of the female petiole

The diagrammatic transverse section of the petiole is irregularly oval to somewhat rectangular in shape, with a highly pubescent margin and shows a discontinuous ring of vascular bundles in the peripheral region of ground tissue. The petiole shows a layer of epidermis with thick cuticle and plenty of simple and glandular trichomes just like that of leaf. Some of the trichomes are composed of broad multicellular lignified basal cells; other trichomes are like those of leaf epidermis but the length of the simple trichomes here in the petiole is very long in comparison to that in the leaf. A total of 5-10 rows of collenchymatous hypodermis lies underneath the epidermis, the remaining ground tissue being parenchymatous, and is traversed with rings of bicollateral vascular bundles.

Surface preparation of female leaves

Surface preparation of female leaves shows few anomocytic stomata, and cicatrix and glandular trichomes in the upper epidermis. Upper epidermal cells are lignified.

Quantitative microscopic characters of the female leaves

The female leaves studied for determining the various leaf constants and general parameters are as follows:

  1. Stomatal index
    1. Upper epidermis, 4.5-5.8
    2. Lower epidermis, 12-13
  2. Vien islet number, 3-4
  3. Palisade ratio, 3.5-4.
Organoleptic characters of the leaves

Organoleptic characters like color and taste of female leaves were also determined. The color of the leaves was green; the taste was slightly bitter and without any characteristic odor.

Physiochemical investigation [11],[12]

The physiochemical constants of female leaves are given in [Table 2].
Table 2: The physiochemical constants of female leaves

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Qualitative chemical tests

The detection of the presence of various constituents like alkaloids, [13] flavonoids, [14] cucurbitacins, [15] resin, etc., was done. Female leaves were subjected to the following preliminary tests. The total alkaloid content was 0.25% w/w.Thin-layer chromatography of alkaloids.

Concentrated methanolic extracts of the leaf was subjected to the chromatographic separation of alkaloids using silica gel G as a stationary phase and toluene:ethylacetate:diethylamine (7:2:1) as a mobile phase. The solvent was allowed to run up to 19 cm. The plate was observed as such under UV light. Rf values of fluorescent spots were calculated, and are given in [Table 3]. The plate was sprayed with Dragendorff's reagent later on and the Rf values of different resolved colored spots were calculated, and are given in [Table 4] and also shown in[Figure 1] [Figure 2].
Figure 2: TLC profile

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Table 3: Observation under long UV light

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Table 4: After spraying Dragendorff's reagent

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High-performance thin-layer chromatography finger printing

One gram of the leaf powder separately was successively macerated three times with 10 ml methanol in a 50-ml stoppered conical flask by keeping it for one night. The pooled extract was evaporated under reduced pressure; the weight of the dried extract was noted down and then it was dissolved in 2 ml of methanol and was subjected to the high-performance thin-layer chromatography (HPTLC) separation [Figure 3].
Figure 3: HPTLC plates

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Gas chromatography

In this, first of all preparative chromatography of the alcoholic (concentrated) extract was done by using a general solvent system of alkaloids and toluene:ethyl acetate:diethylamine (7:2:1). Then the solvent was allowed to run up to an appropriate height; the spots were marked with a needle and silica powder of these spots was carefully scraped and collected separately and dissolved in methanol. The gas chromatogram of the above prepared sample reveals four peaks having retention time 3.79, 14.59, 35.35, and 36.57; the peak corresponding to the sample is the one having retention time of 35.35 and 36.57. Other peaks represent some impurity/contamination of the column. On further analyzing the compound emerging for the GC column in the mass spectrometer, the two probable compounds identified were as follows:

  1. Ferrocene, 1,1", 3, 3'-tetrakis(1,1-dimethylethyl)

    Molecular formula: C 26 H 42 Fe

    Molecular weight: 410
  2. 1,1' 2',1"-Terphenyl,3',4'-dimethyl-5',6' -diphenyl

    Molecular formula: C 32 H 26

    Molecular weight: 410.
Spectral analysis (infrared)

In the spectral study of the newly synthesized (dihydropyridines) molecules, the carbonyl (>C=O) group of the amidic functionality stretching was observed at 1655-1670 cm -1 and another ketonic group (ester, or acetyl) was observed at approximately 1670-1690 cm -1 due to the conjugation with the DHP skeleton. The amide (>C-N stretching) group was observed at 1300-1400 cm -1 .

The stretching of the secondary amine (>NH) group appeared approximately around 3100-3400 cm -1 which indicates the presence of -NH_ group in the compound. Here in almost all the compounds, two -NH sis vibrations are observed since there are two secondary amine groups. One of them shows absorption at lower frequencies due to the carbonyl group attached to it. The stretching C-N appears at 1200-1400 cm -1 which further adds up to the evidence of the presence of the secondary amine group. The T. dioica peak obtain from the infrared spectrum indicates the methyl overtione, amide, aromatic functional groups, etc.


   Results and Discussion Top


For any plant, whether it is dioecious or monoecious, standardization is an essential measure for quality, purity, and sample identification. The microscopic method is one of the simplest and cheapest methods to start with for establishing the correct identity of the source materials. Various exogenous and endogenous factors have been taken into account for determining the quality and purity of a plant. T. dioica Roxb. commonly known as Patoli, Jyotsna, and Patola Patra is one of the highly reputed drugs used in Ayurveda and is incorporated in a number of Ayurvedic formulations, irrespective of gender. Hence, it was thought worth to investigate the pharmacognostic profile of the leaves in the standardization for quality, purity, and sample identification.

 
   References Top

1.Hooker JD. Flora of British India. Reprinted edition, Periodical experts. vol. 2. Delhi; 1973. p 609.  Back to cited text no. 1
    
2.Haines HH. The Botany of Bihar and Orrisa reprinted edition Botanical Survey and India. Vol. 2. Calcutta; 1961. p 406.  Back to cited text no. 2
    
3.Kanjilal VN. Flora of Assam. Reprinted edition, Vol. 2. 1997. p 329.  Back to cited text no. 3
    
4.Shastry KN. Charaka Samhita, Chaukhambha, Vidvybhavan, Varnasi,1970.  Back to cited text no. 4
    
5.Acharya JT. Shusruta Samhita, Chaukhambha Orientalia, Varanasi,1980.  Back to cited text no. 5
    
6.Pandit Sarangdharacharya. Sarangdhar Samhita, with the Commentry Adhanallas Dipika and kasirams Gudhartha Dipika. Eedited by Foot notes by Pandit Parsuram Shastri Vidyasagar, Varanasi; Chaukhambha Oritentalia, 2002.  Back to cited text no. 6
    
7.Chandra Sekar, Mukherjee B, Mukherjee SK. Blood sugar lowering effect of Trichosanthes dioica Roxb. in experimental rat models. Int J Crude Drug Res 1988;26:102-6.  Back to cited text no. 7
    
8.Sharma G, Pandey DN, Pant MC. The biochemical valuation of feeding Trichosanthes dioica Roxb. seeds in normal and mild diabetic human subjects. In relation to lipid profile Indian. J. Physiol. Pharmacol 1990;34:140-8.  Back to cited text no. 8
    
9.Harit M, Rathee PS. The antifungal activity of the unsaponifiable fractious of the fixed oil of Trichosanthes dioica Roxb. Seeds. Asian J Chem 1996;8:180-2.  Back to cited text no. 9
    
10.Harit M, Rathee PS. The antibacterial activity of the unsaponifiable fraction of the fixed oils of Trichosanthes dioica Seeds. Asian J Chem 1995;7:909-11.  Back to cited text no. 10
    
11.The Ayurvedic pharmacopoeia of India, Govt. of India. Ministry of Health and Family Welfare, Department, I.S.M.Q.H. 1 st ed. Part I, Vol.3. 2001.   Back to cited text no. 11
    
12.Pharmacopoeial standards of Ayurvedic formulations. New Delhi: CCRAS; 1987. p. 503, 488.  Back to cited text no. 12
    
13.Stephen KS. Medicinal plant kingdoms. 2 nd ed. Toranto: University of Toranto Press; 1965. p. 15.  Back to cited text no. 13
    
14.Peach K, Tracy MV. Modern Methods of Plant Analysis. Vol. 3. German Edition. Berlin: Springer Verlay; 1955. p. 4, 464.  Back to cited text no. 14
    
15.Egan Sthal. Thin layer chromatography. Berlin, Heidelberg, New York: Springer Verlay; 1969. p. 903 - 904.  Back to cited text no. 15
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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