1. INTRODUCTION:

Portulaca oleracea, (purslane or chickenweed) or referred to as ‘ghol’ in the local Marathi language is a part of the Portulacaceae family. Its characteristic sour taste has been exploited as a dietary source in the form of salad, soup or pickle. The traditional systems of medicine in Asia and Africa have described various pharmacological activities of Purslane. The taxonomic classification of the plant is as follows: Kingdom- Plantae; Order- Caryophyllales; Family- Portulacaceae; Genus – Portulaca; Species- Portulaca oleracea Linn.

P. oleracea prefers temperate climate and is a green annual herb with branched and succulent stems which are horizontal near the base and ascending near the top with a height of 15- 30 centimeters (cm).

The plant water content is over 90 per cent (%)¹. Although the plant may have originated in Asia, it is now found in Africa, Middle East, Europe, North America and Australia. Asian and African traditional systems of medicine indicate that the plant can be used as an anticancer, antidiabetic, hypocholesteremic, neuroprotective, hepatoprotective, nephroprotective, anti-inflammatory, antiulcer, antimicrobial agent. It has been reported that the flavonoid content depends on the plant part.² The different flavonoids present in the plant are kaempferol, myricetin, luteolin, apigenin, quercetin, genistein and display selective cytotoxicity towards human cancer cell lines^1-3. It contains fatty acids such as α- linolenic acid which makes Purslane a rich source of beneficial omega 3 (Ω-3) fatty acids as compared to other vegetables. Recently, a water soluble anionic low molecular weight polysaccharide (gum) has been extracted from the leaves with surface interfacial and emulsification properties and could be used as food emulsifier³. It was reported that the quantity of alkaloids depends mainly on the solvent used for the extraction process. It also contains organic acids and glycosides, cardiac glycosides, anthraquione glycosides with high quantities of vitamin A along with other vitamins such as B complex, C, vitamin E and alanine, catechol, saponins, tannins, steroids, carbohydrates, urea, and minerals such as calcium, iron, copper, phosphorus, manganese and zinc^4-5. Recent studies have reported anti-oxidant activity attributed to the phenolic compounds isolated from P. oleracea crude extract. Apart from the uses described in traditional medicine, experimental evidence points to the plant having neuroprotective activity, anti-cancer activity, anti- ulcer activity, hepatoprotective activity, immune-modulator, nephroprotective activity, anti-diabetic, anti- inflammatory, insecticide and wormicide activities, antimicrobial activity, and antiasthmatic activity^5-7.

Portulaca quadrifida is another species belonging to genus Portulaca, commonly known as chickenweed which grows as a small plant with succulent leaves and woody stem bearing yellow flowers⁸. The taxonomical classification of is as follows: Kingdom- Plantae; Order-Caryophyllales; Family- Portulacaceae; Genus-Portulaca; Species- Portulaca quarifida Linn. Just like P. oleracea, P. quadrifida has been indicated to possess medicinal properties and is used in treatment of asthma, cough, urinary discharges, inflammations and ulcers, haemorrhoids^8-9. These two species of Portulaca are often found growing together as weed and have many active constituents with pharmacological activities such as anti-cancer, anti-hypertensive, anti-diabetic, anti-oxidant, hypolipedemic activity. However, the quality assurance practices followed for crude drug evaluation of these two species of Portulaca were not described in detail. In the present study, the two plant species harvested from Western India were studied for their primary phytochemical evaluation. The parameters studied in this report may be used for quality assurance and primary evaluation of crude drugs from P. oleracea and P. quadrifida.

2. METHODOLOGY:

2.1 Collection of plant material, authentication, extraction:

P. oleracea L. and P. quadrifida L. plants were harvested from a local farm of Walva taluka, District Sangli, State Maharashtra where they were growing as weed. The P. oleracea L. and P. quadrifida L. plant specimens were authenticated by Dr. Dhanaji S. Pawar, Associate Professor, Department of Botany, M. H. Shinde Mahavidylaya, Tisangi, and the plant specimen were deposited with voucher V01 and V04 respectively. Some of the P. oleracea plants were dried in shade and their seeds were separated and stored at 2-8°C for further use.

Figure 1: Image of P. oleraceae L. and P. quadrifida L.

Figure 2: Image of P. oleracea seeds

The plant materials were extracted using Soxhlet extraction and microwave-assisted extraction techniques separately using ethanol, methanol, butanol, and water^10-18. The extracts obtained were dried and stored at 2-8ᵒC.

2.2 Qualitative phytochemical analysis:

The following methods were used for qualitative phytochemical analyses:

· Test for steroids-Salkowski test, Libermann-Burchard test, Libermans’ test

· Test for saponin-Foam test, Hemolytic test

· Test for flavanoids-Shinoda test, lead acetate test, sodium hydroxide test

· Test for alkaloids-Dragendroff’s test, Mayers test, Hagers test, Wagners test

· Test for tannins and phenolic compound-Ferric chloride test, lead acetate test, gelatin test, bromine test, acetic acid test, potassium dichromate test, dilute iodine test, dilute nitric acid test, dilute ammonium hydroxide and potassium ferrocyanide test, ammonium hydroxide and silver nitrate test, potassium permanganate test^19-20.

· Isolation of beta-sitosterol using thin layer chromatography^21-22.

2.3 Physical evaluation:

· Determination of ash values-determination of total ash values, determination of acid soluble ash value

· Determination of extractive values – determination of alcohol soluble extractive value, determination of water- soluble extractive value

· Determination of moisture content^19-20.

2.4 Isolation of beta-sitosterol (β-sitosterol):

The isolation of β-sitosterol from P. oleracea ethanolic extract obtained from microwave-assisted extraction was performed using thin layer chromatography with silica gel G as stationary phase. Chloroform: ethanol (9.5:0.5) mobile phase was used and 5% sulphuric acid in methanol reagent was used for visualization. The obtained retention factor value of the isolated band was compared to the standard β-sitosterol²¹.

β-sitosterol was isolated from P. quadrifida ethanolic extract obtained from microwave-assisted extraction using column chromatography with silica gel G as stationary phase packed in to glass column. Chloroform: ethanol (9.8:0.2) mobile phase was used. The obtained retention factor value of the isolated band was compared to the standard β-sitosterol²¹.

3. RESULTS AND DISCUSSION:

In order to simplify the representation of results, the samples obtained by extraction using Soxhlet extraction and microwave- assisted extraction, separately, will be stated as follows [Table 1]:

Table 3: Results for Qualitative Phytochemical Screening.

Microwave assisted extraction samples

Soxhlet extraction samples

Sr. No.

Phytochemical tested

Method used

Steroids

Salkowski test

Libermann-Burchard test

Libermans' test

Saponin

Foam test

Hemolytic test

Flavanoids

Shinoda test

Lead acetate test

Sodium hydroxide test

Alkaloids

Dragendroff'f test

Mayers test

Hagers test

Wagners test

Tannins and phenolic compounds

Ferric chloride test

Lead acetate test

Gelatin test

Bromine test

Acetic acid test

Potassium dichromate test

Dilute iodine test

Dilute nitric acid

Dilute ammonium hydroxide and potassium ferrocyanide test

Ammonium hydroxide and silver nitrate test

Potassium permanganate test

Presence of a particular phytochemical is represented as (+).

The results indicate presence of all the tested phytochemicals including tannins, flavonoids, alkaloids, saponins, steroids in all the extracts.

3.4 Physical Evaluation:

3.4.1 Microscopic characteristic of Portulaca:

Table 4: Microscopic characteristics physical evaluation of P. oleracea, P. quadrifida

Part used	Transverse section of	Characteristics
Portulaca oleracea	Leaf	Xylem, phloem, parenchyma, epidermal cell, vascular bundle
	Stem	Xylem, phloem, calcium oxalate crystal
	Root	Xylem, phloem, calcium oxalate crystal, starch
Portulaca quadrifida	Leaf	Xylem, phloem, parenchyma, epidermal cell, vascular bundle
	Stem	Xylem, phloem, calcium oxalate crystal
	Root	Xylem, phloem, calcium oxalate crystal, starch

3.4.2 Determination of ash value:

The ash values for P. oleracea dry whole plant, P. quadrifida dry whole plant, P. oleracea seed were 4.3%, 8.2%, and 7.9% respectively. The ash value of P. quadrifida dry whole plant was found to be greater than dried whole plant of P. oleracea and P. oleracea seeds.

3.4.3 Determination of moisture content.

The moisture content determined for P. oleracea fresh whole plant, P. oleracea dry whole plant, P. quadrifida fresh whole plant, P. oleracea seed were 35%, 16%, 30%, and 11% respectively. The moisture content was also the lowest for P. oleracea seed sample.

3.5 Determination of extractive value.

3.5.1 Determination of alcohol soluble extractive value:

The alcohol soluble extractive values for P. oleracea fresh whole plant, P. oleracea dry whole plant, P. quadrifida fresh whole plant, P. oleracea seed were 70 %, 65%, 63%, 78% respectively.

3.5.2 Water soluble extractive value

The water soluble extractive values for P. oleracea fresh whole plant, P. oleracea dry whole plant, P. quadrifida fresh whole plant, P. oleracea seed were 69%, 68%, 62 %, 58% respectively. The alcohol soluble extractive value was greatest for P. oleracea seed sample while P. oleracea fresh whole plant sample exhibited the most water soluble extractive value.

3.6 Isolation of beta-sitosterol (β-sitosterol):

Sample 1 obtained from microwave- assisted extraction was tested for presence of β-sitosterol using thin layer chromatography. The retention factor (R_f) value of β- sitosterol was 0.88 and the reddish brown color band was observed indicating presence of β- sitosterol.

Figure 3: Separation of beta-sitosterol using (a) thin layer chromatography (b) column chromatography.

By column chromatography- Isolation of beta-sitosterol:

Ethanolic extract of P. quadrifida was used and the obtained retention factor of 0.86 was found to be similar to standard beta-sitosterol (Figure 3b).

Earlier report of preliminary phytochemical analysis of ethanolic extract of P. oleracea and P. quadrifida also indicated the presence of pharmacologically important phytochemicals such as tannins, flavanoids, steroids, and saponins^8,22. The percentage yield of extracts indicates the presence of phytochemicals which are selectively more soluble in the respective solvent used and also depend on the method used for extraction. The percentage yield of extracts obtained using microwave- assisted extraction was greater than the corresponding extract obtained using Soxhlet extraction. Also, microwave-assisted extraction was less time consuming as compared to Soxhlet extraction. Reported physicochemical characteristics for P. quadrifida such as total ash value, acid insoluble ash value and water soluble ash value were 9.76, 0.94 and 5.8% respectively⁸.

The extractive value of crude drug indicates the possible extractable phytochemicals in a give solvent and extraction conditions. It also gives an idea of the probable chemical nature of the phytochemicals. The alcohol soluble extractive value was greatest for P. oleracea seed sample while P. oleracea fresh whole plant sample exhibited the most water soluble extractive value. The results indicate that comparatively, P. oleracea seed would have the highest amount of alcohol soluble phytochemicals while P. oleracea fresh whole plant would have the highest amount of water soluble phytochemicals. Other researchers have reported percentage yield of 2.4% and 12% for ethanol and water respectively⁸.

4. CONCLUSION:

The P. oleracea and P. quadrifida extracts contained all the major phytochemical groups as well as β-sitosterol. The extractive values and percentage yield values could be further used by researchers to choose suitable solvent, extraction technique, and as quality indicators to monitor variation in different lots of crude drugs.

5. ACKNOWLEDGEMENT:

The authors would like to thank the Research Directorate, Krishna Institute of Medical Sciences Deemed to be University, Karad, Maharashtra, India for their continued support and encouragement in conducting research.

6. CONTRIBUTION OF AUTHORS:

Sample collection, extraction, quantification and validation experiments were conducted by Trupti Durgawale. Dr. Chitra Khanwelkar reviewed her work. Pratik Durgawale contributed in drafting of the manuscript. All authors have approved the submitted article.

7. DECLARATION OF INTEREST:

None.

8. FINANCIAL ASSISTANCE:

The research work was financially supported by the Krishna Institute of Medical Sciences Deemed to be University, Karad, Maharashtra, India. The said financing body had approved the study protocol after review.

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Received on 08.06.2020 Modified on 04.08.2020

Research J. Pharm. and Tech. 2021; 14(7):3789-3793.

DOI: 10.52711/0974-360X.2021.00656

Sr. No.	Extract	Represented as
1	P. oleracea fresh whole plant ethanolic extract	Sample 1
2	P. oleracea fresh whole plant methanolic extract	Sample 2
3	P. oleracea fresh whole plant butanolic extract	Sample 3
4	P. oleracea fresh whole plant aqueous extract	Sample 4
5	P. oleracea dry whole plant ethanolic extract	Sample 5
6	P. quadrifida fresh whole plant ethanolic extract	Sample 6
7	P.oleracea seed ethanolic extract	Sample 7

Sr. No.	Sample	Percentage yield (%)
Sr. No.	Sample	Soxhlet	Microwave assisted
1	Sample 1	5	7
2	Sample 2	4	6
3	Sample 3	3	5
4	Sample 4	2.5	4.7
5	Sample 5	3	5
6	Sample 6	3.5	6.8
7	Sample 7	6	13