Comparative Standardization of Roots of Boerhaavia diffusa Linn. From Two Different Geographical Regions

 

Arvind Kumar Gupta¹, Kawaljeet Kaur¹, Nawazish Alam², Sayeed Ahmad³, Om Prakash Agarwal¹ and Perwez Alam¹*

¹Natural Product Research Laboratory, Department of Pharmacognosy and Phytochemistry,   SBS College of  Pharmacy, Chuslewal Morh, Patti, (Tarn-Taran), Amritsar, Punjab-143416.

² College of Pharmacy, Jazan University, Saudi Arabia.

³Natural Product Research Lab, Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy,  Jamia Hamdard, New Delhi-110062

Corresponding author: perwez_alam29@rediffmail.com

ABSTRACT:

Boerhaavia diffusa Linn., belonging to family Nyctaginaceae, commonly known as “Punarnava”, is a perennial creeping herb, up to a height of 1-1.2 m. Parts of the plant have been used since long time in the treatment of various diseases. The roots have been reported to possess activities like diuretic, anti-inflammatory, laxative anthelmintic, stomachic, febrifuge, antileprosy, antiscabies, antiviral, and antitumour properties. In the present study, comparative  standardization of the root of B. diffusa from two different geographical regions (Punjab and U.P) have been studied as per the WHO Guidelines to determine the correct identity and purity of the plant part and for the detection of adulteration as well. Botanical authentication and physicochemical parameters gave an idea about the quality of drug. The root powder was extracted with different solvents including Petroleum ether, Benzene, Chloroform, Ethyl acetate, Methanol and Water. Pharmacognostic study (macroscopic and powder microscopic) and physicochemical parameters (extractive values, ash values, foaming index, foreign matter, moisture content) were studied. The extractive values of hot extraction with methanol was found 15.3% w/w and 14.8%w/w and aqueous hot extractive was found 19.3%w/w and 19.7%w/w in Punjab and U.P. regions respectively. Total ash values were found 8.5%w/w and 9.15%w/w in Punjab and U.P. regions respectively. Further phytochemical screening revealed the presence of alkaloids, flavonoids, triterpenoids, phenolic compounds (tannins), saponins and steroids.

 

 

INTRODUCTION:

Medicinal plants are an important therapeutic aid for various ailments. In India from ancient times, different plant parts have been used for the treatment of various diseases¹.Today, there is widespread interest in herbal medicines because herbal medicines are safe, inexpensive and have no adverse effects². B. diffusa Linn. (Nyctaginaceae) commonly known as “Punarnava”, is a perennial creeping herb with a stout root- stock native to India and Brazil³. B. diffusa is a widely grown herbaceous plant all over India, abundant during rainy season⁴.

 

This weed plant occurs throughout the plains and lower hills of India and widely used as a food supplement and Medicinal plants⁵. Its roots were prescribed as a diuretic, analgesic and to improve the functions of kidney and liver⁶ and are also used as a folk medicine for treatment of asthma and abdominal tumor in India⁷. The roots have been reported to possess activities like diuretics⁸, anti-inflammatory⁹, hepatoprotective¹⁰, anticonvulsant¹¹, antifibrinolytic^12-13, adaptogenic¹⁴ and antitumour¹⁵ properties. It is also given for convulsons^16-17. Chemical constituents reported from different parts of B. diffusa are alkaloid punarnavine¹⁸, β-sitosterol, tetracosanoic, hexacosanoic, stearic, palmitic, arachidic acids, β-sitosterol-β-D–glucoside¹⁹, hentriacantane, ursolic acid, and hypoxanthine 9-L-arabinofuranoside²⁰. The present paper describes the comparative Standardization of roots of B. diffusa from two different geographical regions (Punjab and U.P.) as per the WHO guideline²¹.

 

MATERIAL AND METHODS:

Collection, identification and processing of plant materials:

The fresh roots of B. diffusa Linn. were collected from local areas of Amritsar (Punjab) and Allahabad (Uttar Pradesh)  in the month of Aug, 2010 and were identified by the taxonomist, Dr. Saroja Arora, Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar, Ref. No. 0411HRB and the voucher specimen was deposited in Department of Pharmacognosy and Phytochemistry, S.B.S. college of pharmacy patti.

 

Processing of Plant material:

The fresh roots were firstly washed, shade-dried and then powdered and stored in air-dried containers.

 

Pharmacognostic evaluation:

The organoleptic characters including colour, odour, taste, shape and size of root were observed and results are given in Table 1. The powder microscopy of root was also studied after treatment with different reagents prepared according to WHO Guidelines. The figures have been given as Fig.1, Fig.2, Fig.3 and Fig.4.

 

Table.1: Organoleptic Evaluation of  roots of B.diffusa

Characters	Observation
Colour	Cream to Yellowish brown
Odour	Characteristics or Odourless
Taste	Bitter
Shape	Elongated, Fusiform, Tapering
Size	1-4 cm in diameter
Surface	Rough and hard

 

Powder Microscopic characteristics of root of B. diffusa (Punjab)

 

Fig.1: Parenchyma of the Phelloderm

 

Fig.2: A group of Vessels with bordered pits

Powder Microscopic characteristics of root of B. diffusa (U. P.)

 

Fig.3: Fragments of fibers with   parenchyma

 

Fig.4: Stratified Cork Cells

 

Physicochemical evaluation:

The dried roots of B. diffusa Linn. was subjected to standard procedures according to WHO Guidelines for the determination of various physicochemical parameters. The following parameters were determined:

 

Determination of ash values:

The determination of ash values is meant for detecting low-grade drugs, exhausted drugs and sandy or earthy matter. The results of ash values were recorded in Table 2.

 

Total ash value:

Accurately about 3 g of air-dried powder of root of B. diffusa was weighed in a tared silica crucible and incinerated at a temperature not exceeding 450°C until free from carbon, cooled and weighed and then the percentage of total ash with reference to the air-dried powdered drug was calculated. The result of total ash value was recorded in Table 2.

 

Table.2: Different Physicochemical parameters of roots of B. diffusa

Parameters	Punjab region	U.P. region
Total ash (%w/w)	8.5	9.15
Acid insoluble ash (%w/w)	1.5	1.5
Water soluble ash (%w/w)	2.5	3.0
Moisture content (%w/w)	9.0	8.7
Foreign matter (%w/w)	1.5	1.0
Foaming index	166.66	166.66

Table.3: Extractive values (%w/w) of roots of B. diffusa by different extraction method

Solvents	Punjab region			U.P. region
	HE	CM	SE	HE	CM	SE
Petroleum ether	0.9	1.3	1.0	1.0	1.0	1.16
Benzene	1.5	2.0	1.0	1.66	2.33	1.16
Chloroform	2.16	2.66	0.73	2.0	3.0	0.8
Ethyl acetate	3.83	4.0	2.83	3.3	4.33	2.5
Methanol	15.3	13.0	12.2	14.8	12.6	10.7
Water	19.3	15.7	5.83	19.7	15.3	6.7

Abbr.: HE – Hot Extraction, CM – Cold Maceration, SE – Successive Extraction

 

 

 

Table 4:  Phytochemical screening of roots of B. diffusa.

Phytoconstituents	Punjab region			U.P. region
	PEE	ME	AE	PEE	ME	AE
Alkaloids	+	++	+	+	++	+
Carbohydrates	-	+	+	-	+	+
Glycosides	-	+	++	-	+	++
Tannins	-	++	-	-	+	-
Proteins	-	-	-	-	-	-
Saponins	-	+	+++	-	-	+++
Steroids	-	++	-	-	++	-
Flavonoids	-	++	+	-	+	+
Coumarins	-	-	-	-	-	-
Triterpenoids	+	+	-	+	+	-
Lignans	-	++	+	-	+	+

Abbr.:  PEE  :  Petroleum Ether Extract,   ME :  Methanol Extract,   AE  :  Aqueous Extract

+  :  Present,   ++  :   Moderately Present,     +++   :  Strongly Present,   -   :  Absent

 

 

 

Acid insoluble ash value:

The ash obtained in the above method was boiled with 25 ml of dilute HCl for 5 minutes. The residue was collected on ash less filter paper and washed with hot water, ignited, cooled and weighed. The percentage of acid insoluble ash with reference to the air-dried drug was calculated. The result of acid insoluble ash values was recorded in Table 2.

 

Water soluble ash value:

The total ash obtained was boiled with 25 ml of distilled water for 5 minutes. The insoluble matter was collected on an ash less filter paper, washed with hot water and ignited to constant weight at a low temperature. The weight of insoluble matter was subtracted from the weight of total ash. The difference in weight represent the water soluble ash. The percentage of water soluble ash with reference to the air-dried drug was calculated. The result of water soluble ash value was recorded in Table 2.

 

Determination of moisture content (LOD):

The powdered drug sample (3 g) was placed on a tared evaporating dish and dried at 105°C for 4-5 h and weighed. The drying was continued until two successive readings matched each other or the difference between two successive weighing was not more than 0.25% of constant weight. The result was recorded in Table 2.

 

Determination of Foreign matter:

A 500 g of the plant material was spread in a thin layer and the foreign matter was sorted into groups by visual inspection and using a hand lens. The remainder of the sample was sifted through a no. 250 sieve; dust was regarded as mineral admixture. The sorted foreign matter was weighed. The content of each group was calculated in grams per 100 g of air dried sample. The result was recorded in Table 2

 

Determination of Foaming Index:

About 1 g of the powdered sample was transferred to a conical flask containing 100 ml of boiling water. Decoction was prepared and filtered. The decoction was poured into 10 stoppered test tubes (height 16 cm, diameter 16mm) in successive portions of 1 ml, 2 ml, 3 ml etc. up to 10 ml and the volume of liquid in each test tube was adjusted to 10 ml with water. The test tubes were stoppered and shaken in a lengthwise motion for 15 seconds. The test tubes were allowed to stand for 15 minutes and the height of the foam was measured. The results are assessed as follows:

·         If the height of the foam in every tube is less than 1 cm, the foaming index is less than 100.

·         If a height of foam of 1 cm is measured in any tube, the volume of the decoction in this tube is used to measure the foaming index.

·         If the height of foam is more than 1 cm in every tube, the foaming index is over 1000.

The result was recorded in Table 2.

 

Determination of extractive values:

The air-dried, accurately weighed drug was treated with different solvents: petroleum ether, benzene, chloroform, ethyl acetate, methanol and water and the extractive values were determined by using different methods including Hot extraction, Cold maceration and Successive extraction. The results were recorded in Table 3.

Table.5: Fluorescence analysis of root B. diffusa (Punjab)

S. No.	Solvents/Reagents	Visible light	Short UV (254 nm)	Long UV (366 nm)
1.	Drug powder as such	Yell.          brown	Light brown	Dark brown
2.	Drug + conc. H2SO4	Brown	Gr.  brown	Bl. Brown
3.	Drug + conc. H2SO4 + Distilled water	Brown	Dark brown	Black
4.	Drug + conc. HCl	Light brown	Gr. brown	Black
5.	Drug + conc. HCl + Distilled water	Yell. brown	Gr.  brown	Bl. Brown
6.	Drug + conc. HNO3	Yell. brown	Gr. brown	Black
7.	Drug + conc. HNO3 + Distilled water	Yell. brown	Gr. brown	Black
8.	Drug + Methanol	Light brown	Bl. brown	Br.  Black
9.	Drug + Chloroform	Gr. brown	Dark brown	Black
10.	Drug + Petroleum ether	Brown	Bl. brown	Br. Black
11.	Drug + FeCl3	Yell. brown	Gr. brown	Black
12.	Drug + Picric acid	Brown	Dark brown	Black
13.	Drug + 10% NaOH solution	Yell. brown	Gr. brown	Br. Black
14.	Drug + NH3 solution	Light brown	Gr. brown	Black
15.	Drug + Acetic acid	Yell. brown	Gr. brown	Br. Black
16.	Drug + Distilled water	Brown	Dark brown	Br.  Black

 

 

 

Table 6: Flourescence analysis of root of B. diffusa (U.P.)

S. No.	Solvents/Reagents	Visible light	Short UV (254 nm)	Long UV (366 nm)
1.	Drug powder as such	Yell. brown	Light brown	Black
2.	Drug + conc. H2SO4	Brown	Gr. brown	Bl. Brown
3.	Drug + conc. H2SO4 + Distilled water	Brown	Dark brown	Black
4.	Drug + conc. HCl	Brown	Dark brown	Br.  Black
5.	Drug + conc. HCl + Distilled water	Dark brown	Gr. brown	Bl.  Brown
6.	Drug + conc. HNO3	Yell. brown	Brown	Black
7.	Drug + conc. HNO3 + Distilled water	Yell. brown	Gr. brown	Black
8.	Drug + Methanol	Light brown	Bl. brown	Br.  Black
9.	Drug + Chloroform	Light brown	Dark brown	Black
10.	Drug + Petroleum ether	Brown	Dark brown	Br.  Black
11.	Drug + FeCl3	Yell. brown	Brown	Black
12.	Drug + Picric acid	Yell. brown	Brown	Black
13.	Drug +10% NaOH	Brown	Bl. brown	Br.  Black
14.	Drug + NH3 solution	Yell. brown	Gr. brown	Bl.  Brown
15.	Drug + Acetic acid	Yell. brown	Gr. brown	Br.  Black
16.	Drug + Distilled water	Gr. brown	Dark brown	Br.  Black

Abbr. Gr.- Greenish,      Yell.- Yellowish,  Br.- Brownish,     Bl.- Blackish

 

 

 

Phytochemical screening:

Preliminary phytochemical screening of the various extracts with different reagents and solvents was performed and the results were recorded in Table 4.

 

Fluorescence analysis:

The powdered drug was examined under ordinary light, short UV(254nm) and long UV(366nm) light using different solvents. The results were recorded in Table 5 and Table 6.

 

RESULTS AND DISCUSSION:

The root powder was extracted with different solvents including Petroleum ether, Benzene, Chloroform, Ethyl acetate, Methanol and Water. Pharmacognostic study (macroscopic and powder microscopic; shown in fig.1, 2, 3, 4 respectively) and physicochemical parameters (extractive values, ash values, foaming index, foreign matter, moisture content) were studied. The extractive values of hot extraction with methanol was found 15.3% w/w and 14.8%w/w and aqueous hot extractive was found 19.3%w/w and 19.7%w/w in Punjab and U.P. regions respectively. Total ash values were found 8.5%w/w and 9.15%w/w in Punjab and U.P. regions respectively. Moisture content was found to be 9.0% and 8.7% in Punjab and U.P. regions respectively. Foreign matters were found to be 1.5% and 1 % in Punjab and U.P. regions respectively. The foaming index was found to be 166.6 in both Punjab and U.P. regions. The phytochemical screening showed the presence of alkaloids, glycosides, saponins, steroids and lignans in the both regions.

 

CONCLUSION:

Generated data can be used for determining correct identity and purity of plants part and detection of adulteration as well. Botanical authentication and physicochemical parameters will give an idea about the quality of drug. All these parameters, which are being reported, could be useful in identification of distinctive features of the drug. From the phytochemical study, it can be concluded that only selected species of Boerhaavia contains the reported phytoconstituents. Hence, detailed screening may be done to isolate the active constituent so that it may be scientifically proved to access the pharmacological responses of the plant to ascertain it’s folklore use.

 

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Received on 25.10.2011          Modified on 12.11.2011

Accepted on 20.11.2011         © RJPT All right reserved