Pharmacognostic, Physicochemical Standardization and PhytochemicalAnalysis of leaves of Hydrolea zeylanica Vahl. (Hydrophyllaceae).

 

Mohammad Shamim Qureshi¹*, A. Venkateshwar Reddy², G.S. Kumar³ and Lubna Nousheen²

¹R & D cell, Jawaharlal Nehru Technological University, Kukatpally Hyderabad, - 50035, Telangana, India

²Anwarul Uloom College of Pharmacy, New Mallepally, Hyderabad – 500001, Telangana, India

³School of Pharmacy, International Medical University, Bukit Jallil, Kuala Lumpur 57000. Malaysia

 

ABSTRACT:

The most important aspect of Pharmacognosy is to evaluate the quality and purity of crude drugs by means of various parameters.For the quality and purity of medicinal plants, pharmacognostic, physicochemical and preliminary phytochemical studies of the leaves of Hydrolea zeylanica Vahl, family Hydrophyllaceae. were carried out.The macroscopic and microscopic assessment revealed characters that are of diagnostic value and useful in authentication of the plant. The Physicochemical analyses reveals values for moisture content, water soluble extractive, alcohol soluble extractive and total ash which are within the World Health Organisation (WHO) standards for crude drug from medicinal plants. Phytochemical studies for secondary metabolites revealed the presence of Glycosides, volatile oils, tannins, carbohydrate, sterols, flavonoids and alkaloids are present in the extractsInformation obtained from these studies can be used as markers in the standardization and identification of this plant as a herbal remedy and also towards monograph development on the plant.

 

 

 

INTRODUCTION:

Plants have formed the sophisticated traditional medicinal systems that have been in existence for thousands years ^[1-2]. The medicinal use of plants as medicines is dated back to early man^[3]. They constitute an effective and major source of traditional and modern medicines and play an important role in health care programs.Pharmacognosy is a simple and reliable tool for obtaining the complete information regarding crude drugs^[4-7].

 

Today with the present surge of interest in the phyto-therapeutics, the availability of authentic plant material is becoming scarce. Since crude drugs form the basis for the manufacture of numerous medicinal preparations, accurate determination of drug identity forms an essential part of its study. It becomes most important to make an effort for standardization of the plant material as medicine. The process of standardization can be achieved by stepwise pharmacognostic studies^[8].

 

Hydrolea zeylanica (L) Vahl, family Hydrophyllaceae found throughout India in moist and swampy places^[9].It is an annual herb with procumbent and branching stems up to 30 cm. long. The leaves are dark green, narrow and pointed at the tip and are arranged alternately on the swollen, spongy stems. The stems growing above water are firmer and sturdier^[10]. It is also known as Koliary and used for anthelmintic activity^[11]. However, no pharmacognostic study has been carried out on its leaves part and hence the objective of the present study is to evaluate various pharmacognostic features including macro and microscopic and physicochemical characterization of the leaves of Hydrolea zeylanica.

 

MATERIALS AND METHODS:

Collection of plant material

The leaves of Hydrolea zeylanica (L) belonging to the family Hydrophyllaceae were collected from local area of Chittoor district (India) and was identified and authentified by Dr. K. Madhava Chetty, Plant taxonomist (IAAT:357), Department of Botany, Sri Venkateswara University Tirupati. A.P. India. The plant bearing voucher No. 1012 (10/12/2014) was deposited at Department of botany Sri Venkateswara University Tirupati, A.P.

 

Macromorphological Description

The leaves of Hydrolea zeylanica (L) were subjected to macroscopic studies which include organoleptic characteristics. This can be done by means of the sense organs, which provide the simplest as well as quickest means to establish the purity and identity to ensure quality of a particular drug. Organoleptic characters such as size, shape, colour, odour, taste and fracture of stem bark, leaf structure like margin, apex, base surface, venation and inflorescence, etc. are evaluated.These parameters are considered as quite useful in quality control of the crude drug and were evaluated as per standard WHO guidelines^[12-14].

 

Microscopic characteristics

The microscopic study is done by taking an appropriate section (leaf) of the plant under study. Each distinguishing character can be noted down, some of which are retained in the powder study also. Some of the chemicals which are used in obtaining sections are phloroglucinol, chloral hydrate, safranine, methyl orange, etc.Fresh leaves of Hydrolea zeylanica(L) were selected for the microscopical studies. Microscopic sections were cut by free hand sectioning. Numerous temporary and permanent mounts of the microscopical sections of the leaf specimen were made and examined under the microscope. Histochemical reactions were applied with staining reagents on transverse sections by reported methods ^[15-16].Photomicrographs of the microscopical sections were taken with the help of Digital Microscope (MOTIC) provided with MOTIC IMAGE PLUS 2.0 software.

 

Powder study

Preliminary examination and behaviour of the crude powder with various chemical reagents and microscopical examination was carried out as per reported methods^[17-18].Powder study is similar to microscopic study except here dried powder is taken instead of various section of the plant. The dried small pieces of leaves were powdered by means of pistil and mortar and electric grinder. The physical characters of powdered drug that are colour, smell and taste were studied.

 

Physicochemical Evaluation

Estimation of Physicochemical constants of the powder leaf has been carried out to evaluate the quality and purity of the drug. Various physicochemical factors like foreign organic matters, moisture contents, ash values and extractive values were calculated as per WHO guidelines. The information collected from these test was beneficial to obtain quality standards and for standardization^[19-20].The factors which are calculated are moisture content, loss on drying, total ash, acid-insoluble ash, alcohol and water-soluble extractive values, ethyl acetate soluble extractive value, petroleum ether soluble extractive value, acetone soluble extractive value, etc. Ash values are used to determine quality and purity of drug.It indicates presence of various impurities like oxalate, carbonate and silicate. The water soluble ash is used to evaluate the amount of inorganic compound present in drugs. The acid insoluble ash provides mainly silica and indicates contamination with earthy material. Moisture content of drugs must be at minimal level to prevent the growth of bacteria, yeast or fungi during storage. Analysis of extractive values determines the amount of active constituents in a given amount of plant material when extracted with a particular solvent. The extractions of any crude drug with a particular solvent yield a solution containing various phytoconstituents. The compositions of these phytoconstituents rely upon the nature of the drug and solvent used. It also confirms whether the crude drug is exhausted or not^[21].

 

Florescence Analysis

Many herbs show fluorescence when the cut surface or powder is exposed to various UV light and this can be useful in their identification. The fluorescence character of the leaf powders (40 mesh) was studied both in UV light and daylight (366nm and 254nm) and after treatment with different reagents like hydrochloric acid, sodium hydroxide, nitric acid and ferric chloride etc^[22].A small quantity of dry leaf powder is placed on oil free clean microscopic slide and 1-2 drops of freshly prepared reagent solution is added, mixed by gentle tilting the slide and wait for few minutes. Then the slide is kept inside the UV chamber and observe the colour in visible light, short (254 nm) and long (365 nm) ultra violet radiations. The colour observed by application of different reagents in different radiations is recorded^[23].Usually the changes in colours arenoted in reagents like Powder + 1 N NaOH (alc), Powder + 1 N NaOH (aq), Powder + Ammonia, Powder + Picric acid, Powder + 50% HCl, Powder + Petroleum ether, Powder + 50% H₂SO₄, Powder + Ethyl acetate, Powder + Ethyl alcohol, Powder + Methanol, etc. Some constituents exhibit fluorescence in the visible range in daylight. The ultra violet light produces fluorescence in many natural products which do not visibly fluoresce in daylight. If substance themselves are not fluorescent, they may usually convert into fluorescent derivatives or decomposition products by applying different reagents. Hence crude drugs are often assessed qualitatively in this way and it is an important factor for pharmacognostic evaluation of crude drugs^[24].

 

Phytochemical Investigations

The qualitative chemical tests carried out for the identification of the different nature phytoconstituents present in the powered crude drugs by standard procedures.They are usually tested for the presence of alkaloids, flavonoids, phenols, tannins, cardiac glycosides, triterpenes, steroids and saponins^[25-26].

 

RESULT AND DISCUSSION:

Macromorphologcal Description

Hydrolea zeylanica is an erect to prostrate, perennial herb, usuallybranched and growing about 10 - 60cm tall. The organoleptic studies of the leaf estimated greyish black in colour, with characteristic odour and astringent taste.Figure 1 and 2 explains the extra features observed in leaf. The results of macromorphology were depicted in Table 2 and 3.

 

A - Twig, B - Flower, C and D - Stamens,

E – Pistill, F – T.S. of Ovary.

 

Table 1.Hydrolea zeylanica

 

Table 2: Organoleptic Characters

 

 

Table 3: Quantitative Macromorphology

 

Microscopic evaluation of leaf

The leaf of Hydrolea zeylanica is short petioled, lanceolate, rather obtuse, smooth, bright green above, below marked with numerous prominent parallel veins.The lamina and midrib were less differentiated from each other. Midrib is thick, it has epidermal layer of small hemispherical cells with heavy cuticle. The vascular bundle of the midrib was single, large and elliptical comprising of a wide horizontal band of xylem and a broad zone of phloem. The xylem consists of small, thick walled vessels and fibres. The vascular bundle was enclosed by wide parenchyma zone both on the adaxial and abaxial sides. In between the epidermal layers and were seen four or five layers of thick walled compact parenchyma cells (Figure 3).

 

Figure 3. TS of leaf through midrib with lamina. [Ads- Adaxial side, Cu- Cuticle, Ep- Epidermis, LP- Lower palisade cells, Ph- phloem, SM- spongy mesophyll, UP- Upper palisade cells].

 

The lamina has thickreticulate veins and veinlets. The primary and secondary veins branch profusely and give rise to ultimate veinlets. The vein islets were distinct, small and squarish or rectangular. Each vein islet has one or two vein terminations. The terminations were unbranched or branched once. The vein-terminations have a cluster of large, lobed sclereids called terminal sclereids (Figure 4).

 

Figure 4. Vein-islets and Vein-termination. [VI- Vein islets, VT- Vein termination].

 

Trichomes are glandular or eglandular, abundant all along margins of both surfaces. Trichomes of three types: sessile glands, globular or dome shaped, few in number, found along upper epidermis, contain dense reddish contents; uni or biseriate Trichomes, sparsely distributed, without conducting tissue; and multiseriate  glandular Trichomes or digestive glands, abundant, consists of a stalk of variable length with a large globose head (Figure5).  

 

 

Figure 5. Types of Trichome

 

Powder characteristics

The powder was characterised on its different morphological features. The dried fine powder was stained with chloral hydrate to detect the presence of calcium oxalate crystals. They were prismatic in nature. When stained with phloroglucinol and concentrated hydrochloric acid (HCl) the vascular bundles and lignified fibres were observed.The fibres were either straight or curved at one end or both ends (Figure 6-7).

 

 

Figure6.Calcium Oxalate Prisms	Figure 7. Lignified Fibres

 

 

Physicochemical Parameters

The results of the various physicochemical constants ofraw material lie within the limit which is given in Table 4; this signifies that the purity and quality of raw material was good enough.the results of foreign organic matter indicates the presence of any organism, part or product of an organism, other than that named in the specification and description of the crude drugs concerned; which was found to be 0.33 ±0.27, it indicates that there may be present of product  or part of an organism in very less amount.Insufficient drying may lead to spoilage by moulds and bacteria and makes possible the enzymatic destruction of active principles.Not only the ultimate dryness of the drug is important, equally important is the rate at which the moisture is removed and the condition under which it is removed thus the determination of moisture content also provide the method of preparation of drug; ^[27-28]and it is observed that the moisture content of the drug was found to be 03.86 ±0.72 which signify that the drug is properly dried and properly stored. The total ash is particularly important in the evaluation of purity of drugs, i.e. the presence or absence of foreign matter such as metallic salts or silica^[29].An analytical result for total ash was found to be 10.41 ±1.26.The amount of acid-insoluble matter present was 2.12±1.31. The water soluble ash was found to be 5.26±1.16; this parameter is used to detect the presence of material exhausted by water. As the ash values of the crude drugs lies within the fair limit which signify its quality and purity and gives idea about the total inorganic content. The water soluble extractive values indicated the presence of sugar, acids and inorganic compounds^[30].The water soluble extractive value found to be 10.82±1.18 and the alcohol soluble extractive was found to be 4.03±1.12 which signify that the large amount of constituents of leaves was soluble in water and alcohol.

 

Table 4: Physicochemical parameters

Values are expressed as mean ± SD of six values.

 

Fluorescence analysis

The fluorescence analysis of the powder drug was done and results are given in Table No. 5.The powder was treated with various reagents and the mixture was observed under UV light (254 nm).Fluorescence study is an essential parameter for first line standardization of crude drug. In fluorescence the fluorescent light is always of greater wavelength than the exciting light. Light rich in short wavelengths is very active in producing fluorescence and for this reason ultraviolet light produces fluorescence in many substances which do not visibly fluoresce in daylight^[31].

 

 

 

 

Table 5: Fluorescence Analysis of Leaf Extract of Hydrolea zeylanica.

S. No.	Chemical Test	Hydrolea zeylanicaDay light	Hydrolea zeylanicaUV light
1	Sample as such	Green	Dark Green
2	Powder + 1N aq. NaoH	Yellow	Green
3	Powder + 1N alc. NaOH	Yellow green	Dark Green
4	Powder + 1 N HCl.	Greenish Yellow	Green
5	Powder + 50% HNO3	Pale Green	Dark Green
6	Powder + 50% H2SO4	Pale Yellow	Green
7	Powder + Methanol	Green	Blackish Green
8	Powder + NH3	White	Green
9	Powder + I2	Reddish Orange	Yellowish Brown
10	Powder + FeCl3	Brownish Orange	Dark Brown

 

 

 

Preliminary Phytochemical Screening

Chemical tests were carried out on all the extracts for the qualitative determination of phytochemical constituents. Glycosides, volatile oils, tannins, carbohydrate, sterols, flavonoids and alkaloids are present in the extracts and the results were shown in Table No.6.

 

 

Table 6.Preliminary phytochemical screening of different extracts of leaf of Hydrolea zeylanica.

Note: (+++) Present (---) Absent

 

 

CONCLUSION:

The present study may be useful to supplement the information with regard to its identification and standardization. Standardization is an essential measure for purity and quality, and sample identification. Macromorphology and microscopy along with the quantitative analytical microscopy is one of the cheapest and simplest methods to start with for establishing the correct identity of the materials. Physiochemical and phytochemical analysis of leaf confirm the quality and purity of plant and its identification. Here the information collected was useful for further pharmacological and therapeutical evaluation along with the standardization of plant material.

 

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Received on 28.11.2016          Modified on 18.12.2016

Accepted on 22.12.2016      ©A&V Publications All right reserved

Res.  J. Pharmacognosy and Phytochem. 2017; 9(1): 01-07.

DOI: 10.5958/0975-4385.2017.00001.2