INTRODUCTION:

Plant latex is an aqueous milk-like secretion, stored in the vacuole of highly specialized elongated laticifer cells and it immediately oozes out once the plant tissues gets damaged.¹ The latex comprises a remarkable range of organic compounds such as lipids, rubbers, resins, carbohydrates, terpenoids, alkaloids and cardiac glycosides. It also contains various proteins and enzymes such as proteases, chitinases, and glucosidase.

Recently, research has led to understanding the existence, function and structural elucidation of naturally derived secondary metabolites in the laticifers.²

Euphorbia is the largest genus in the Euphorbiaceae family and incorporates over 2000 species, which are mostly characterized by presence of milky white latex. The latex of representative species was found to be toxic and extremely irritating to the skin.¹

Euphorbia fusiformis Buch.-Ham.ex D.Don (Euphorbiaceae) is a rare, perennial, succulent, acaulescent, scapigerous herbs grown up to 20 cm to 25 cm. Its occurrence in India is limited to few localities of Maharashtra, Uttar Pradesh, Nagar Haveli, Telangana, Andhra Pradesh, Karnataka, Tamil Nadu and West Bengal.^3,4,5

Traditionally, local tribal people of India had been using Euphorbia fusiformis plant to treat headache, arthritis, gout, paralysis, diarrhoea, abdominal diseases, abdominal tumor, liver disorders, urinary stones, chronic wound cracks, skin disease, eczema and poor lactation.³ In scorpion and snake bites, plant latex was used as an antidote.⁶ The latex had been used to treat liver disorders, diarrhoea, chronic wounds and skin diseases. It is helpful for burning off warts and eczema.^6,7.8.

In spite of the versatile traditional uses of Euphorbia fusiformis there is no scientific report available for the preliminary phytochemical investigation and acute oral toxicity study of latex. With this background, we have conducted preliminary phytochemical analysis and acute oral toxicity study of Euphorbia fusiformis latex to provide a base for further exploration with respect to isolation of constituents and evaluation of pharmacological activities.

EXPERIMENTAL PROCEDURE:

Collection of latex:

The fresh tubers of Euphorbia fusiformis were collected from the local region of Satara, Maharashtra, India in rainy season. The plant material was authenticated by Botanical Survey of India, Pune. Cuts were made on the roots with a sharp knife and oozing whitish latex was collected in amber coloured bottle. The latex was stored in deep freezer below -4⁰C till further use.

Physicochemical characterization of latex:⁹

The latex of Euphorbia fusiformis was subjected to following physicochemical parameters:

Refractive Index:

The refractive index of latex in distilled water (1:1) was measured using a refractometer (Make-Abbe, model-SSU).

Density (weight per mL):

As per Indian Pharmacopoeia method, weight per mL of latex was estimated by a pycnometer.

Percent solid content:

5mL latex was transferred in preweighed petridish and dried up to constant weight in oven at 110⁰C. Upon drying the percentage of dry weight was measured.

pH:

Calibrated pH meter (Make - Toshcon Industries, model - CL46+) was used for measurement of pH of the latex.

Total resinous matter:

10mL latex was fractionated with 10 percent ethanol (20mL each) three times. Separated and concentrated layer of ethanol (25mL) forms flocculated precipitate when equivalent volume of distilled water was added. The precipitate of total resinous matter was collected, dried and measured.

Table 1: Physico-chemical parameters of latex

Parameter	Value
Refractive Index	1.35 ± 0.11
Weight per mL	1.08 ± 0.05 g
Percent solid content	14.93 %
pH	5.4 ± 0.12
Resinous Matter	12.56 %

Solubility study:^9,10

Latex was dissolved in solvents of different polarity and solubility was observed.

Table 2: Solubility study of latex

Solvent Used	Solubility
Water	Soluble*
Ethanol	Soluble
Methanol	Soluble
Acetone	Sparingly soluble
Chloroform	Sparingly soluble
Ethyl acetate	Sparingly soluble
n-Hexane	Freely soluble

*Turbid solution

Preliminary phytochemical investigation:

Phytochemical screening of ethanolic solution of Euphorbia fusiformis latex was performed to detect the presence of different classes of phytoconstituents by standard methods. The qualitative results are expressed as (+) for the presence and (-) for the absence of phytoconstituents.

Carbohydrates^11,12 (Molisch’s test):

Two drops of Molisch’s reagent was mixed with 2mL of the latex solution. Few drops of concentrated Sulphuric acid were added slowly from the side of the test tube. Appearance of the violet ring at the junction indicates presence of carbohydrates.

Proteins^11,13 (Biuret test):

2mL of biuret reagent was added to 3mL of the latex solution. Appearance of violet or pink color indicates presence of proteins.

Test for resins¹⁴:

1mL of latex solution was treated with a few drops of acetic anhydride solution followed by 1mL of Concentrated Sulphuric acid. Coloration ranging from orange to yellow indicates presence of resins.

Fats and oils¹⁵ (Spot test):

A drop of fresh latex was pressed between filter paper. Appearance of permanent stain on filter paper indicates presence of the fats and oils.

Volatile oils¹⁶ (Stain test):

A drop of fresh latex was pressed between filter paper. Observation of temporary stain on filter paper indicates the presence of the volatile oils.

Steroids⁹(Liebermann test):

Chloroform solution of latex was mixed with 3mL of acetic anhydride. The mixture was heated, cooled, and 3-4 drops of concentrated Sulphuric acid was added. Formation of brown precipitate indicates the presence of steroids.

Steroids⁹(Liebermann Burchard tets):

To the chloroform solution of latex, 1-2mL of acetic anhydride was added and mixed. To this, two drops of concentrated sulphuric acid was added from side of the test tube. Formation of brown ring at junction of two layers indicates the presence of steroids.

Terpenoids⁹ (Salkowski test):

Chloroform solution of latex when treated with an equal volume of concentrated sulphuric acid. A red to purple colour formation indicates the presence of terpenoids.

Cardiac glycosides^14,16 (Killer–Killani test):

To the latex solution 1mL of glacial acetic acid and 2-3 drops of 5% Ferric Chloride solution was added. To this, 2ml of concentrated Sulphuric acid was added slowly from the sides of the test tube. The formation of a reddish brown color at the junction and bluish green color to upper layer indicates the presence of the cardiac glycosides.

Anthraquinone glycosides¹⁶ (Modified Borntrager’s test):

2mL of latex solution was mixed with 2mL of 5% ferric chloride solution and 2mL of 10% hydrochloric acid solution. The mixture was boiled in water bath for 5 min. and filtered while hot, after cooling, it is extracted with 5mL of chloroform. The chloroform layer after treatment with equal volume of dilute ammonia solution shows pinkish red color which indicates presence of anthraquinone glycosides.

Cynogenic glycosides¹⁷:

Latex in ethanol was mixed with equal volume of cold concentrated sulphuric acid. Formation of intense colour indicates the presence of cynogenic glycosides.

Coumarin glycosides¹⁶(Fluorescence test):

Latex in ethanol was treated with 5mL of dilute sodium hydroxide. Appearance of blue or green fluorescence indicates presence of coumarin glycosides.

Saponins¹⁶(Foam test):

1mL of latex in ethanol was diluted 20 times with distilled water in a graduated cylinder and shaken for 15 min. Formation of foam of about 1cm layer indicates presence of saponins.

Flavonoids¹⁶ (Shinoda test):

Latex was dissolved in ethanol and heated. About 0.5g of magnesium metal and 4-5drops of concentrated hydrochloric acid was added. Formation of red or orange color indicates presence of flavonoids.

Alkaloids¹⁶ (Mayer’s test):

2mL of latex solution, 4–5 drops of Mayer’s reagent was added. The formation of a cream colored precipitate indicates the presences of alkaloids.

Tannins and Phenols¹⁶ (Ferric chloride test): about 0.5g of latex was mixed with 10mL of distilled water and filtered. To 2mL filtrate, 2mL 5% Ferric chloride solution was added. The formation of black or blue‑green coloration or precipitate indicates presence of tannins.

Table 3: Phytochemical investigation of latex of E. fusiformis

Phytochemicals	Test	Latex
Carbohydrates	Molisch’s test	−
Proteins	Biuret test	−
Resins	Acetic anhydride test	+
Fats and Oils	Spot test	−
Volatile oils	Stain test	−
Steroids	Liebermann test	+
Terpenoids	Salkowski test	+
Cardiac glycosides	Killer–Killiani test	−
Anthraquinone glycosides	Modified Borntrager’s test	−
Cynogenic glycosides	Sodium picrate test	+
Coumarin glycosides	Fluorescence test	−
Saponins	Foam test	+
Flavonoids	Shinoda test	−
Alkaloids	Mayer’s test	+
Tannins and phenols	Ferric chloride test	−

+: Present, −: Absent

THIN LAYER CHROMATOGRAPHY^18-21:

TLC plates (Make: Merck, Type- 60 GF254) were used for chromatography study. The solvents of analytical grade were purchased from Sigma-Aldrich for TLC profiling.

Solution of latex in n-hexane was spotted on thin-layer silica gel plates and then developed using mobile phase methanol: n-hexane (9:1). After activation, the plates were visualized using long wave ultraviolet light and retention factor (Rf) values were calculated for each band observed. Two distinct spots were observed with Rf value 0.3 and 0.6.

Latex solution in ethanol was spotted on thin-layer silica gel plates and developed using mobile phase ethyl acetate: n-hexane (1:1). The plates when visualized using long wave ultraviolet light one distinct spot was observed with retention factor (Rf) value of 0.5.

ACUTE ORAL TOXICITY:^22-27

Acute toxicity study was performed to ensure the safety profile of the latex.

Experimental animals:

Female Swiss albino mice (25-35g) were used for the study. They were procured from National Institute of Biosciences, Pune, India. All the animals were kept under standard laboratory conditions (Temperature: 22±3°C; 40%-60% relative humidity; 12 h light/12 h dark cycle) and acclimatized for one week before commencement of the experiment in an animal house approved by Committee for the Purpose of Control and Supervision of Experiments on Animals, New Delhi, India. They were feeded standard diet supplied by Nutrivet Life Sciences, Pune. The animals were fed regularly and water ad libitum.

The study protocol was approved by Institutional Animal Ethics Committee of MAEER’s Maharashtra Institute of Pharmacy, Pune, India (Approval No: MIP/IAEC/2015-2016/M1/Appr/005).

Procedure:

Acute oral toxicity study was performed by acute toxic class method of OECD 423 guideline. Briefly, mice were randomly divided into three group containing three animals in each. They were fasted for 4 h with free access to drinking water. The fasted mice were treated with a single dose of latex (2000mg/kg body weight). Latex was suspended in 0.5% w/v carboxy methyl cellulose solution and given once by oral route (5ml/kg body weight). The control group received vehicle, i.e. 0.5% w/v carboxy methyl cellulose solution (5ml/kg) once by oral route. After treatment, mice were closely observed for 4 hours, periodically during the 24 hours, and daily thereafter for a total period of 14 days. Number of mortality were recorded during observation period. Clinical signs of toxicity such as changes in skin fur, eyes and mucous membranes, diarrhoea, salivation, sleep and lethargy were monitored. The gross behaviours like body positions, locomotion, rearing, tremors and gait were observed. The effect on stereotype, vocalization, body weight and intake were also recorded.

The single oral administration of Euphorbia fusiformis latex did not produce mortality in mice by the dose of 2000mg/kg body weight furthermore, there was no clinical and behavioural signs of toxicity in Euphorbia fusiformis latex-treated mice when compared against control mice

RESULTS AND DISCUSSION:

Result of this study indicates the presence of alkaloids, glycosides, terpenoids, steroids and saponins in Euphorbia fusiformis latex. From acute oral toxicity study single dose LD₅₀ was found to be more than 2,000 mg/kg. The acute oral toxicity study provides preliminary data on the toxicity profile of latex of Euphorbia fusiformis. The latex of Euphorbia fusiformis is safe when administered orally and therefore characterized as Category 5 in the GHS system.

CONCLUSION:

The present work reports the physicochemical evaluation, phytochemical investigation and acute oral toxicity study of latex of Euphorbia fusiformis for the first time. The results obtained from above studies will provide a platform for further exploration with respect to isolation of constituents, formulation studies and pharmacological evaluation for validating its traditional claims.

ACKNOWLEDGEMENTS:

The authors are thankful to the Principal and management of MAEER’s Maharashtra Institute of Pharmacy, Pune, Maharashtra, India for their encouragement and availing the internet and laboratory facilities for this research work.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 27.10.2020 Modified on 22.04.2021

Research J. Pharm.and Tech 2022; 15(3):1123-1127.

DOI: 10.52711/0974-360X.2022.00188