INTRODUCTION:

Medicinal plants are the most active part of traditional medicines for the treatment of various diseases. However, a key obstacle, which inhibits the promotion of the use of herbal medicines in developed countries, is the lack of evidence in the documentation and the absence of stringent quality control measures. There is a need for a record of all the research work carried out on traditional medicines in the form of documentation.

Due to these reasons, standardization of the plant and plant parts used in herbal medicinal preparations is extremely important. Different techniques and methodologies such as pharmacognostic and phytochemical studies can be used for the process of standardization of herbal drugs. These methods are helpful in preventing the adulteration of plant material used for the preparation of herbal medicines by confirming their authenticity. Raw material standardization ensures the reproducible quality of herbal medicine which will help us to justify its safety and efficacy. For this purpose, we have done pharmacognostic studies of Hibiscus hispidissimus Grifitth belongs to the family Malvaceae. The sour leaves are used as food ingredient and used in the preparation of South Indian cuisine. The leaves are the source of an ayurvedic drug ‘Sathambasthi’. This drug is a major constituent of pancamlatailam (one of the five acidic drugs), an oil preparation for body anointing. The leaves are also an ingredient in the Ayurvedic drugs Annabhedi sinduram and Abhram. The leaves have anti-inflammatory and anthelmintic activities. Tribal healers of Kerala region use this plant to treat Liver diseases. It is said to improve digestion and also has anthelmintic action. The juice of the leaves is mixed with honey and used in the treatment of eye diseases. In summer the roots of the plant are infused with water which is used as a cooling drink. Decoction of the root bark is used as a remedy for poisons, swellings, and cleansing of kidney^3,4. Since there is no published work regarding the pharmacognostic parameters of this highly useful medicinal plant, which is essential for its standardization. Considering the ethnomedicinal value of Hibiscus hispidissimus Griffith, the present work has been taken up to document the pharmacognostic parameters of leaves and stems of this plant.

MATERIALS AND METHODS:

Plant collection and authentication:

The plant specimens (leaves and stems of Hibiscus hispidissimus Griffith) for the proposed study were collected from Palode, Thiruvananthapuram district of Kerala in August. The collected plant specimens were examined and authenticated by Dr. E.A. Siril, HOD, Department of Botany, University of Kerala, Karyavattom, Thiruvananthapuram. A voucher specimen (Voucher No. KUBH 11158) has been deposited in the Herbarium of the Department of Botany for future reference.

Macroscopical studies^{4, 5}:

The morphological characters of leaves and stems were studied and the results were tabulated.

Microscopical study of fresh leaves stems^{4, 5}:

The handmade transverse sections of leaves and stems were taken using a sharp blade and stained with various staining reagents. The stained sections were observed under Labomed compound microscope.

Powder microscopy⁵:

Determination of leaf constants⁶:

The leaf constants such as stomatal number, stomatal index, vein islet number, and vein-termination number were determined by microscopic method with the help of Camera Lucida.

Physico-chemical Analysis^{5, 7, 8}:

Physico-chemical parameters such as moisture content, total ash, water soluble ash, acid insoluble ash, water soluble extractive, and alcohol soluble extractive were carried out according to WHO guidelines for the standardization of herbal drugs.

Loss on drying:

About 5g of the accurately weighed drug was taken in a tared evaporating dish. The sample was placed in hot air oven at 105°C for 5hrs and weighed. The process was continued i.e., the drying and weighing at one-hour intervals until the difference between two successive weighing corresponds to not more than 0.25%. The constant weight is noted when two consecutive weighs after drying for 30min and cooling for 30min in a desiccator shown not more than 0.01g difference.

Total ash:

Incinerated about 3g accurately weighed powdered drug in a tarred platinum crucible at a temperature not exceeding 450ºC (in muffle furnace) until free from carbon. Cooled and weighed, repeated to get constant value. The percentage of total ash was calculated with reference to the air-dried drug.

Water soluble ash:

The ash obtained from total ash procedure was boiled with 25ml water and insoluble matter was collected in a tarred crucible on ashless filter paper. Then, it was washed with hot water and ignited for 15min at a temperature not exceeding 450ºC. Subtracted the weight of insoluble matter from the weight of ash, the difference in weight represents water soluble ash value with reference to the air-dried drug.

Acid Insoluble ash:

The total ash obtained was boiled with 25 ml of 2 M HCl for 5 minutes. The insoluble matter was collected on an ashless filter paper (Whatmann 41), and washed with hot water. Transferred the filter paper containing insoluble matter to the tarred crucible, ignited, weighed and calculated the percentage acid-insoluble ash value with reference to the air-dried drug.

Sulphated ash value:

Heated a platinum crucible to redness for 10 minutes, allowed it to cool in a desiccator, and weighed. Put 3g of the powdered drug, accurately weighed, into the crucible, and ignited gently at first, until the substance is thoroughly charred. Cooled, moistened the residue with 1 ml sulphuric acid, heated gently until white fumes no longer evolved, and ignited at 800 ± 25°C until all black particles have disappeared. The crucible was cooled, and a few drops of sulphuric acid was added and again heated. The ignition was carried out as before, cooled, and weighed to get a constant weight. Calculated the percentage of sulphated ash with reference to the air-dried drug.

Extractive values:

Macerated 5 g of the air-dried, coarsely powdered drug (passed through sieve no.18 and retained in sieve no.60) with 100 ml of solvent (Distilled water, methanol, diethyl ether) in a closed flask for 24 hours, shaken frequently during the first 6 hours and allowed to stand for remaining 18 hours. Then it was filtered, without any loss of solvent, evaporated 25 ml of the filtrate to dryness in a tarred flat-bottomed shallow dish and dried at 105ºC, to constant weight. Calculated the percentage of extractive value with reference to air-dried drug.

Determination of crude fiber content^{5, 6}:

Weighed 2 g of powdered drug in a beaker and added 50 ml of 10% v/v nitric acid and heated to boil with constant stirring (till about 30 minutes after boiling starts). Then strained through fine cotton cloth on a Buchner funnel. Washed residue with boiling water and transferred the residue from the cloth to the beaker. Added 50ml of 2.5% v/v sodium hydroxide solution and heated to boil. Maintained at boiling point for 30 minutes with constant stirring. Strained and washed with hot water as mentioned earlier. For quantitative determinations, transferred the residue to a cleaned and dried crucible. Weighed the residue and determined the percentage of crude fiber content.

Fluorescence analysis:

The powdered leaves and stems of Hibiscus hispidissimus Griffith were taken on a clean glass slide and treated with the freshly prepared reagent. Mixed gently and observed under daylight, and UV light (254 nm and 365 nm) to assess the fluorescent behavior of the drug, and the results were recorded and tabulated.

RESULTS AND DISCUSSION:

Macroscopical studies:

Macroscopical studies of leaves and stems are depicted in Table no.1 and Fig no.1.

Table1. Macroscopical evaluation of leaves and stems of H.hispidissimus

Sl.No	Features	Observations
Leaves of H.hispidissimus
1	Shape	Simple, Palmate or broadly ovate, 3 – 5 lobed
2	Size	3-10 × 3-8.5 cm
3	Color	Green
4	Odour	Characteristic
5	Taste	Sour
6	Leaf arrangement	Alternate
7	Venation	Palmate
8	Apex	Accuminate
9	Margin	Serrate
10	Base	Cordate or truncate
11	Petiole	2-8 cm long
12	Texture	Hairy and prickly on nerves beneath
Stems of H.hispidissimus
13	Habit	Rambling or climbing
14	Colour	Reddish brown
15	Texture	Woody and covered with hooked prickles

Figure 1(a). Leaves of H.hispidissimus Figure 1(b). Stems of H.hispidissimus

Microscopical studies:

Handmade transverse sections of leaves and stems were stained with safranin and phloroglucinol-HCl reagent. Then observed under the microscope. The details of microscopical studies are shown in Figures 2 & 3.

Figure 3 (b). T.S of Hibiscus hispidissimus Griffith leaf stained with safranin observed under magnification 10x. a-trichomes, b-upper epidermis, c- upper collenchyma, d- upper palisade cells, e- xylem, f-phloem, g- lower collenchyma h- lower epidermis.

Powder microscopy:

Fine powder of Hibiscus hispidissimus Griffith leaves and stems were stained with phloroglucinol-HCl reagent and observed under magnification 10x. The results are depicted in the following figure 4.

(a) (b) (c)

(d) (e)

Figure 4. a. Xylem vessel, b. Spiral vessel, c. Phloem fiber, d. Raphides, e. Covering trichomes

Determination of leaf constants

Table 2. Leaf constants of Hibiscus hispidissimus Griffitth

Sl.No.	Parameters	Range
1	Stomatal number	11-14
2	Stomatal index	28-34.14
3	Vein islet number	10-11
4	Vein termination number	10-12

Figure 5 (a). Venation

Figure 5 (b). Paracytic stomata on lower surface of leaf

Determination of physico chemical parameters:

Table 3: Physico chemical parameters of coarse powder Hibiscus hispidissimus Griffith leaves and stems.

Sl. No	Physico chemical constants	Average (%w/w)
Ash values
1	Total ash value	6.55 ± 0.0003
2	Acid insoluble ash value	1.00 ± 0.0002
3	Water soluble ash value	4.30 ± 0.0025
4	Sulphated ash value	3.30 ± 0.0032
Extractive values
5	Water soluble extractive value	27.46 ± 0.0012
6	Alcohol soluble extractive value	19.20 ± 0.0005
7	Ether soluble extractive value	2.67 ± 0.0007
8	Loss on drying	5.56 ± 0.0022
9	Crude fiber content	29.50 ± 0.0132

All values are expressed in mean ± SEM, n = 3

From Table no.3, it is clear that the total ash, water soluble ash, acid insoluble ash, and sulphated ash are lesser in amount. It indicates that the amount of inorganic matter and non-physiological matter present in the plant material is too less. So, we can conclude that the plant material is not adulterated.

Table 4: Fluorescence analysis of powdered leaves and stems of Hibiscus hispidissimus Griffith

Sl. No:	Treatment	Daylight	UV 254nm	UV 365nm
1.	Dry powder	Yellowish green	Green	Light green
2.	Powder + 1N HCl	Brownish-yellow	Green	Light yellow
3.	Powder + Alcoholic KOH	Green	Yellowish green	Light Yellow
4.	Powder +Picric acid	Greenish yellow	Green	Black
5.	Powder + 5% FeCl₃	Black	Dark green	Olive green
6.	Powder + Glacial acetic acid	Golden green	Green	Yellow
7.	Powder + Acetone	Yellow-green	Light green	Orange
8.	Powder + Ethanol	Yellow-green	Green	Light orange
9.	Powder + ConH₂SO₄	Black	Greenish black	Light yellow
10.	Powder + Con HCl	Olive green	Olive green	Light green
11.	Powder + ConHNO₃	Reddish brown	Green	Reddish brown
12.	Powder + Dil. NH₃	Green	Dark green	Green

Extractive value gives an idea about the amount of active constituents present in a specified amount of crude drug when extracted with a particular solvent. From the data (Table 3) obtained, water soluble extractive value is highest followed by alcohol and ether soluble extractive value.

Loss on drying indicates the moisture content present in the crude drug. It was only 5.56% (from Table 3), which does not encourage the growth of microorganisms.

The crude fiber content determined can be used as a measure of the nutritive value of the drug⁶.

Fluorescence analysis:

The powdered leaves and stems show various fluorescence on examination under daylight, UV 254 and 365 nm after treatment with various reagents (Table.4). This property of the drug can be used as a part of its preliminary phytochemical profile.

CONCLUSION:

Literature review revealed that not many studies are done on the leaves and stems of Hibiscus hispidissimus Griffith, still claiming various traditional medicinal uses. The present study focused on its pharmacognostic studies. Both macroscopic and microscopic studies were done. Physico chemical characters such as ash value, extractive value, moisture content, and crude fiber content were also determined. All the pharmacognostic parameters evaluated in this study are useful in determining the identity, purity, and quality of the drug. So, this study will help in the standardization of this highly usable medicinal plant.

CONFLICTS OF INTEREST:

The Authors have no conflicts of interest regarding this study.

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Received on 27.04.2023 Modified on 19.05.2023

Res. J. Pharmacognosy and Phytochem. 2023; 15(3):215-219.

DOI: 10.52711/0975-4385.2023.00033