Pharmacognostical Investigation of Trapa natans L Leaves

 

Chandana Majee¹*, Rupa Mazumder¹, Alka N. Choudhary²

¹Department of Pharmaceutical Chemistry, Noida Institute of Engineering and Technology (Pharmacy Institute), Knowledge Park II, Greater Noida, Uttar Pradesh, India 201306.

²Department of Pharmaceutical Chemistry, Shri Guru Ram Rai University, Dehrakhas, Patel Nagar,

Dehradun, Uttarakhand, India 248001.

 

ABSTRACT:

Background: Trapa natans L., is annual aquatic plant generally kwon as Water caltrp, Water chest nut belonging to the Trapaceae or Lytraceae family. Trapa natans L is use for the treatment of wide no of diseases without proper standardization. Objective: To give the right pharmacognostical and photochemical information of the Trapanatan L leaves. In this study pharmacognostical investigation of the fresh leaves and powder drug were done to determine the macroscopical, microscopical, quantitative physicochemical and phytochemical property of the drugs. Method: Macroscopical, quantitative and qualitative microscopy, physicochemical evaluation, extractive value, florescence analysis and phytochemical analysis were done according to the WHO guideline. Result: Macroscopical analysis showed that, leaves are greenish to purplish color, rhomboidal shape; alternate, acute, margin is dentate, pinnate venation. Microscopic evaluation showed that leaf is dorsi ventral in nature, upper layer epidermis cells were covered with cuticle layer. Single layer of barrel shape cell were present bellow the upper epidermis layer. Trichomes are generally multicellular. Anomocytic stomata were observed in upper epidermis. From the experiment it was found that methanolic extract give the highest extractive value. Phytochemical analysis gives the evidence for the presence of carbohydrate, alkaloids, glycoside, steroids, flavonoids, tannin, and triterpenoids. Qualitative phytochemical analysis give the evidence for presence of high amount total phenolic content. Conclusion: Different pharmacognostical parameters assessed in this examination help to detection and standardization of Trapa natans L., leaves.

 

 

 

INTRODUCTION:

Nature provide abundant number of plants and has possible all solution for human disease. Science the ancient times nature is the back bone of the health care system. It is unfeasible to find any plant which has no therapeutic value^1,2. It is cleared that the natural products play the major role in the drug development system. Although uses of synthetic medicine is growing due to the cost, quick action, easy to evaluation but there was a big question mark about their safety profile^3,4. Now a day 80% of the developing country’s people rely on the natural products as the most reliable sources for medicines⁵.

 

Trapa natans L., is the very significant medicinal plant belong to the family Trapace or Lythraceae. Trapa natans L., is also kwon as Water Caltrop or Water Chest Nut. T natans is the free-floating plant, grow in ponds and lake in slightly acidic condition and in sunny position⁶. The chemical compounds cycloeucalenol and ursolic acid were isolated from this pant. The Trapa natans L., fruits contain high amounts of minerals such as calcium, phosphate, iron, magnesium, and manganese. The inner part of the fruits also contains vitamin A, vitamin B, vitamin C, riboflavin, and nicotinic acid. This plants also contain high amounts of antioxidants such as phenol, flavoin and flavonoids^7,8. In Ayurvedic framework Trapa natans L., is a vital plant, use in the stomach problems, genitourinary system diseases, liver disorder, kidney damage and so on. From the various literature survey it was found this plant also use for fiver, inflammation, diarrhea, and leprosy. Various pharmacological investigation give the evidence that T. Natans posse analgesics activity, anti-diabetic activity, immunomodulatory activity and nortropic activity⁹.

 

From the review of literature it can conclude that no systematic pharmacognostical investigation was done on the leaf of the Tarpa natans L., Therefore this present study deals with macroscopical, microscopical and phytochemical investigation of Trapa natans L leaf, which will be treat as reference standard for identification, characterization authentication of the plant from the adulterant’s product.

 

MATERIALS AND METHODS:

Trapa natans L., plants were collected from Purulia district, West Bengal, in November 2018.Trapa natans L., was identified and authenticated by Dr. Anjula Pandey, Principal Scientist, National Bureau of Plants Genetic Resource (NBPGR), New Delhi.

 

Macroscopy:

Plant was collected, wash and cleaned properly with water and shade dried and kept for the farther investigation. The fallowing macroscopical investigation of the plant leaves were noted for example color, order, flavor, size, apex, margin, venation, presence or absence of petiole, base surface, lamina, texture and so on¹⁰

 

Microscopy:

Leaf microscopy:

Transverse section of leaf was stained by the safranine and phloroglucinol according to the standard method and observed under Trinocular Microscope. Photos were capture by camera lucida for common and specific character.

 

Powder microscopy:

Powder microscopy is useful for further pharmacological and therapeutic evaluation along with the standardization of plant material. In powder microscopic examination powder drug was treated with different reagents like Phloroglucinol, Conc. HCl, Ruthenium red after that powder materials ware observed under the microscope¹¹.

 

Physicochemical Analysis:

The epidermal strips of leaves were subjected to the qualitative microscopy to determine the palisade ratio, stomatal index, stomatal number, vein termination number, vain islet number. The other physical parameter of the powder drug such as moisture content, total ash, acid insoluble ash, water- soluble ash and loss of drying values were determine as per WHO guideline¹².

 

Fluorescence analysis:

Fluorescence analysis of powdered drug were done by placing the dry powder leaf on a slide and treated with various chemical reagents and then observed by the UV and Visible light. The developed colour were noted within 1-2 minutes to avoid drying^13,14.

 

Extractive value with different solvents:

Kwon quantity of powder drug was taken extracted with different solvent (nonpolar to polar) like petroleum ether, chloroform ethyl acetate, ethanol, and methanol. After that filtrate found from various solvent were evaporated and weight of the extracts were taken. Percentage of the extractive values percentage were determined in regard to initial weight of the dried powder drug. Which solvent was given the highest extractive value will subjected for the farther investigation¹⁵

 

Phytochemical analysis: For phytochemical screening various solvent extract were taken and treated with the various chemical reagent. Given below in the Table 3¹⁶.

 

Quantitative phytochemical estimation:

Total phenolic content determination:

The total phenolic content of Trapa natans L., was estimates by the Folin-Ciocalteu assay method. Total phenolic content was determine by diluting the crude extract with suitable solvent to attain the dilution range 0.0-100μg of the galic acid/ml. The prepares extract mix with 1ml of distilled water and 250μl of Folin-Ciocalteu reagent. After that 2.5ml of 7% Na₂CO₃ solution added to it and allowed to stand for 6 minutes. Absorption of the sample was measured after 90 minutes by UV-VIS spectroscopy. By fallowing the method standard solution of gallic acid was prepared. Standard curve of gallic acid prepared to determine the concentration of phenolic content of the extract. Total phenolic content express as mg of GAE/g dry weight¹⁷.

 

Determination of Total flavonoid:

Total Flavonoids content was estimate determine by extracting 10gm of powder drug with 100ml 80% methanol water. The obtained extract was filtrate through the What man filter paper (No 42) The whole. Filtrate transferred into crucible and evaporated until constant weight obtained. Flavonoid’s content of Trapa natans L., leaves extract was determine by the aluminum chloride colorimetric assay method and express as mg Chrysin equivalents (CE)/ 100 g dry weight¹⁸.

 

Determination of Total alkaloid determination

Total alkaloids contents of Trapa natans L., extract measured by Bromocresol green colorimetric assay. The Total alkaloids content of the extracts was  expressed as percentage of  atropine equivalent per 100 gm  dry weight of sample.¹⁹

 

RESULTS:

Macroscopy:

From the microscopical evaluation it was found that leaves are dark greenish color and beneath part is radish purple, characteristic odor and slight bitter. Leaves are Rhomboid Shape, 2-6 cm long. Base and apex of leaves is acute, generally dentate margin, and pinnate venation. Arrangements of leaves are rosette type.

 

Microscopic analysis of Trapa natans L leaves:

Leaf microscopy:

Trapa natans L., leaves are dorsiventral leaf. It is divided into two parts lamina and midrib.

 

A. Lamina:

The lamina region of the leaf consists of upper epidermis, lower epidermis and mesophyll. Lower epidermis and upper epidermis can be distinguished. Lower epidermis and upper epidermis both are consisting of single layer of barrel shape cell which are compactly arranged and having a thin wall. Both the upper and lower epidermis cell covered by thin cuticle layer. The stomata present in upper layer of epidermis due to the free floating nature of leaves. Multicellular trichomes present in the lower epidermis. Calcium oxalate crystal observed in the spongy region. The spongy tissues are composed of two layer of spongy tissue. The large numbers of air cavity present in the spongy region. The air cavity separated by a unilayer cells. This air cavity helps the leaf for floating. In each vascular bundle phloem and xylem present but xylem is very poorly developed.

 

B. Midrib:

The midrib region is covered with parenchyma sheath protected by collenchyma cell. The vascular system is not well developed. The midrib consists of phloem and xylem. Xylem vessel present toward the center portion where the phloem vessel present toward the periphery portion. Many starch grain also found in this region. Fig.1

 

Figure 1: T.S of Trapa natans L Leaves (UE- upper epidermis, LE- lower epidermis, AC – air chamber Ph – phloem; PC – parenchyma cell Ph – phloem; Pi – pith; Xy– xylem Co-calcium oxalate, Cu–cuticle layer)

 

Powder microscopy:

Powder was dark green color with character odor. Form the microscopic observation of powder drug it was found that T. natans L., leaves contain anomocytic stomata, multicellular trichomes. Prismatic calcium oxalate crystal presence in the powder drug. Rectangular lignified fiber and cork cell were also present. Shown in the Fig.2.

 

Figure 2 - Powder Microscopic Characters of Trapa natans L., leaf (Anomocytic Stomata, B. Lignified Fibers, C. Calcium Oxalate, D. Multi cellular covering trichomes, E. Epidermis Cell, F. Cork cell)

 

Quantitative microscopy:

In the quantitative microscopic stomatal number, vin-islet number, vain termination number of the leaf were determine and given in the Table1.

 

Table 1: Vital quantitative microscopy of Trapa natans L leaf

Stomatal number	Vin- islet number	Vein termination number
19.4±0.14	8.6±0.094	10.4±0.248

Mean± standard deviation where numbers of n= 5

 

Physicochemical evaluation:

Physicochemical parameter:

Determination of ash values give the idea about earthy matter and inorganic substance present in the drug and also suggest about the impurities present in the drug. Fig.3

 

Figure 3: Physicochemical parameter of Trapa natans L leaf ( n=5)

 

Fluorescence analysis:

Florescence analysis of the powder drug were determine by treating the powder drug with deferent chemical reagents and observed under UV and visible light. Fluorescence analysis result are given in Table 2

 

Table 2: Fluorescence Characteristic of Powder Leaves

 

Extractive value determination:

Extractive value of powder drug were determine with different solvent given in Fig.4

 

Phytochemical screening:

By taking extracts obtained by using different solvents, various phytochemical tests were performed to determine the active ingredient present in the plant extracts. Maximum no of active ingredients were founds from the methanolic extract (Table 3).

 

Figure 4- Extractive values of Trapa natans L., leaves in different solvents

 

Table 3- Phytochemical Screening of various solvent extract for the presence of active chemical constituents of Trapanatans L

Plant constituents	Petroleum ether	Benzene	Chloroform	Ethyl acetate	Acetone	Methanol	Aqueous
Amino acids	-	-	-	+	+	-	-
Alkaloids	-	+	+	-	-	-	-
Steroids	-	-	+	+	-	+	+
Triterpenoids	+	+	-	+	-	+	+
Saponins	-	-	+	+	+	+	+
Flavanoids	-	-	-	-	+	+	-
Tannins	-	-	-	+	+	+	+
Glycosides	-	-	-	-	+	+	-
Carbohydrates	+	-	-	+	-	+	+

 

Quantitative phytochemical estimation

Table 4-Quantitave estimation of photochemical of levees of Trapa natans L

Values represented as mean ± standard error (n=6)

 

DISCUSSION:

Plant’s kingdom serve as the biggest source medicine for the treatment of various types of disease. But, it the biggest task to identify the correct plant. It is very essential to avoid the duplicity of the plant material before starting any research. So, the first priority for work with the herbal drug is authentication and standardization of the plant material. Trapa natans L., currently used for the treatment of various types of diseases without proper standardization. Standardization of the herbal drug is very important before adding the drugs in the herbal pharmacopeia. Microscopical and microscopically evaluation help to identification of Trapa natans L., plant. Pinnate venation generally found in monocot plant. Cuticle layers on the upper epidermis is a barrier which helps to reduce the evaporation rate of water from the leaves tissue²⁰. Calcium oxalates are common mineral generally present in the higher plant. Calcium found from the environment biologically synthesize to calcium oxalate²¹_. The shape of the calcium oxalate varies plant to plant according to the genetic code. The function of calcium oxalate is to control ionic balance and osmosis²². Presence, absence, position and structure of the trichomes are used for identification of plant. Trichomes reduce the water evaporation, bringing down the temperature; reflect the radiation of light²³.

 

Ash value is important parameter for standardization of herbal drug. High ash value indicate the present of adulteration²⁴. Acid soluble ash give the indication for the presence of silica. Water soluble ash value indicate the water soluble material or water soluble impurities²⁵. From the experiment the total ash, water soluble, acid insoluble ash and sulphated ash were found respectively 10.2±0.95, 4.14±0.30, 2.37±0.25 and 6.26±0.56.

 

Extractive values give the idea about the selection of solvent for farther experiment. In this experiment petroleum ether, benzene, chloroform, ethyl acetate ethanol and methanol were used for determination of extractive value of Trapa natans L., leaves. By using Methanol maximum extractive value was found. Qualitative phytochemical test give the evident for the presence of high amount of phenolic compounds.

 

The fluorescent analysis of powdered drug plays the very significant role for the determination of quality and purity of the drug. Fluorescent studies of powdered drug are also importance to distinguish the different varieties of the plant and adulteration.²⁶ Due to the various chemical component present in the powder extract the ultraviolet light or visible light give the different shading. Numbers of compounds exhibit fluorescence in UV light like alkaloids which is not feasible in visible light²⁷. From the Phytochemical analysis it was revealed that maximum phytoconstituent present in the extract obtained from methanol.

 

ACKNOWLEDGEMENT:

The authors are thankful to Dr. Avijit Mazumdar Director of NIET Pharmacy Institute, AKTU University of Uttar Pradesh, for his support and appreciation throughout the work.

 

CONFLICT OF INTEREST:

Author has no conflict of interest.

 

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Received on 22.06.2020           Modified on 02.09.2020

Accepted on 07.10.2020         © RJPT All right reserved