Pharmacognostical and Antimicrobial Studies of the Stem Barks of Prosopis cineraria (L) Druce

 

Stellaa Robertson1*and Narayanan N2

1Dept. of Pharmacognosy, Maharaji College of Pharmacy, Besant Nagar, Chennai 600 090, Tamil Nadu, India.

2College of Pharmacy, Madras Medical College, Chennai 3, Tamil Nadu, India.

 

ABSTRACT

Prosopis cineraria (L) Druce is one of the highly valued plant in the Indigenous System of Medicine. Its bark is said to be a potent drug for several ailments such as leprosy, dysentery, bronchitis, asthma, leucoderma, piles, muscular tremors, asthma, rheumatism and inflammations. It is also known to possess anthelmintic, antibacterial, antifungal, antiviral and anticancer activities. In view of its medicinal importance, the present research was focused on the pharmacognostical and antimicrobial properties of stem bark of P. cineraria by in vitro approach. Pharmacognostic investigation of the fresh, powdered and anatomical sections of the stem barks of P. cineraria was carried out to determine its macroscopical and microscopical characters along with the physico-chemical and preliminary phytochemical analysis. The antimicrobial activity of ethyl acetate and hydro alcoholic extracts of stem barks were evaluated against two Gram positive (Staphylococcus aureus, Staphylococcus epidermidis), two Gram negative (Escherichia coli, Klebsiella pneumoniae) bacterial strains and two fungal strains (Aspergillus niger, Aspergillus fumigatus) by agar disc diffusion method. Minimum Inhibitory Concentration (MIC) required for cessation of microbial growth was evaluated by agar streak dilution method. Both the extracts showed dose dependent activity against the microorganisms investigated. The hydroalcoholic extract exhibited significant activity against the test organisms than the ethyl acetate extract.

 

KEYWORDS: Prosopis cineraria, Antibacterial, Antifungal, Minimum Inhibitory Concentration (MIC), Stem bark.

 

INTRODUCTION:

Prosopis cineraria (L) Druce syn: Prosopis spicigera Linn., and Mimosa cineraria Linn1 belongs to the family Leguminosae, sub-family Mimosoideae and Class Magnoliopsida Dicotyledons, is a large shrub up to 10m height with branches prickly, prickles curved and compressed. The species is found throughout India extending to Persia2. It is known as Vanni or jambu in Tamil; Jand or Khejra in Hindi and Shami in Sanskrit3,4. The whole plant is used in Indigenous System of Medicine (ISM) and is called Kalpa Plant in Ayurveda and Siddha literature. The literature survey revealed that the bark of the tree have been used in leprosy, dysentery, bronchitis, asthma, leucoderma, muscular tremors and rheumatism. It is also known to possess anthelmintic, antibacterial, antifungal, antiviral and anticancer activities. Water-soluble extract of the residue from methanol extract of the stem bark exhibits anti-inflammatory properties5,6,7,8. The decoction of the bark in combination with the barks of Erythrina indica and Azadirachta indica is used in syphilis9. Stem bark contains vitamin K, n-octacosyl acetate, n-hexacosanoic acid and n-octacosanoic acid. Alcoholic extract of stem barlso yielded glucose, rhamnose, sucrose and starch10. Inspite of the numerous medicinal uses attributed to this plant, there is no pharmacognostical report required for the quality control of the crude drug. Hence the present study includes the morphological and anatomical evaluation, determination of physico-


Table 1: Analytical parameters of the stem bark of P. cineraria

 

 

S. no.

Characteristics

Results (% w/w)

I.

Ash values

1.

Total ash

13.93

2.

Water soluble ash

2.65

3.

Acid insoluble ash

3.42

4.

Sulphated ash

18.40

 

II.

 

Extractive values

1.

Ethanol soluble extractive value

0.21

2.

Water soluble extractive value

1.87

 

III.

 

Loss on drying

 

12.16

 

Table 2: Preliminary phytochemical studies of extracts of P. cineraria

S. No.

Tests

EAPC

HAPC

1.

Alkaloids

+

+

2.

Carbohydrates

-

-

3.

Glycosides

-

-

4.

Phenolic compound

+

+

5.

Tannins

-

-

6.

Proteins and amino acids

-

+

7.

Saponins

+

+

8.

Gums and mucilages

-

-

9.

Phytosterol

-

+

10.

Fixed oils and Fats

-

-

11.

Flavonoids

+

+

+ = Present; - = Absent

 

chemical constants and preliminary phytochemical analysis of the different extracts of P. cineraria.

 

The study also includes the antimicrobial screening of the extracts (ethyl acetate and hydroalcoholic extracts) of P. cineraria which could be useful for the development of new tools as antimicrobial agents for the control of infectious diseases.

 

Fig 1 Stem bark of P. cineraria

 

MATERIALS AND METHODS:

Plant material:

The plant specimens of P. cineraria were collected from the Mylapore, Thiruvallur district, Tamil Nadu, India in the month of September 2007. The specimens were positively identified and authenticated by Prof. P. Jayaraman, Director of Plant Anatomy Research Centre, West Tambaram, Chennai. Voucher specimen (No: A-43/PARC) has been deposited in the same Institution.

 

Macroscopic and microscopic analysis:

Macroscopical features such as shape, size, fracture, colour, odour and taste were carried out according to the method of Evans11. Microscopical studies were also carried out using Nikon Labphot-2 microscope units. For normal observations, bright field was used. For the study of crystals and lignified cells, polarized light were employed. Since these structures have birefringent property under polarized light, they appear bright dark background. The sections of the bark were taken through TS, TLS and RLS with the help of rotary microtome. The average thickness of the sections was 10-12 m. Dewaxing of the sections was done and the sections were stained out according to the methods outlined by Brain and Turner12 and Johansen13.

 

Physico-chemical analysis:

The physico-chemical values such as the percentage of ash values, loss on drying and extractive values were performed according to official methods prescribed (Indian Pharmacopoeia)14 and the WHO guidelines on quality control methods for medicinal plant materials (WHO/QCMMPM guidelines)15.

 

Preparation of extracts:

The stem barks were collected, shade dried and coarsely powdered by using pulvarizer. These coarse powders were then extracted with ethyl acetate (EAPC) and 50% alcohol (HAPC) by cold percolation process to yield the respective extracts. The extracts were reduced to a molten mass by rotary vacuum evaporator and the respective yields of EAPC and HAPC were 0.87% w/w and 1.16% w/w respectively.

 

Preliminary phytochemical screening:

The preliminary phytochemical screening was carried out by using standard procedure described by Kokate16 and Harborne17.

 

Fig 2 T.S of bark through periderm

 

Antimicrobial activity study:

Bacterial and fungal strains:

The microbes used to determine the antimicrobial activity are two Gram positive (Staphylococcus aureus ATCC 6538P, Staphylococcus epidermidis ATCC 155), two Gram negative (Escherichia coli ATCC 8739, Klebsiella pneumoniae ATCC 29665) bacterial strains and two fungi (Aspergillus niger ATCC 16404, Aspergillus fumigatus ATCC 13073). All the bacterial and fungal cultures were procured from the Institute of Microbial Technology, IMTECH, Chandigarh, India.

 

Screening of Antimicrobial Activity

The antimicrobial screening was performed by agar diffusion method using a paper disc18,19. Nutrient agar and Saborauds dextrose agar media were used for the antimicrobial screening. 1ml suspension of the microorganisms (matched with McFarland barium sulphate standard) was inoculated with 100ml of the

 

 


Table 3: Zone of Inhibition of stem bark of P. cineraria

S. no.

Organisms

EAPC

HAPC

Standard

(mm)

50 g

100 g

200 g

Standard

(mm)

50 g

100 g

200 g

1.

Staphylococcus aureus

31

14

18

26

30

19

25

32

2.

Staphylococcus epidermidis

30

14

17

25

30

20

22

29

3.

Escherichia coli

31

15

18

20

30

16

20

29

4.

Klebsiella pneumoniae

29

12

15

19

31

15

20

27

5.

Aspergillus niger

32

12

19

21

28

14

16

18

6.

Aspergillus fumigatus

31

14

19

22

27

14

18

20

 


 

Fig 3 T.S of bark through Secondary phloem

 

Fig 4 Collapsed phloem- sclerotic bands & crystals

 

sterilized (autoclaved at 120˚C for 30 min) medium (40-50˚C). The paper impregnated with the extracts (50, 100 and 200 g/ml) was placed on the solidified medium. The plates were preincubated for 1h at room temperature and incubated at 37˚C for 24h and 48h for antibacterial and antifungal activities respectively. Ciprofloxacin (50g/disc) and Ketaconazole (50g/disc) was used as standard for antibacterial and antifungal activity respectively.

 

Fig 5 TLS of Phloem- PLM view

 

The Minimum Inhibitory Concentration (MIC) for the above organisms was found by agar streak dilution method20. Stock solutions of the extracts (EAPC and HAPC) were mixed with the known quantity of molten sterile agar media aseptically to provide the required concentrations. About 20 ml of the media containing the extract was poured into each sterile petridish and allowed to solidify. Microorganisms were then streaked one by one on the agar plate aseptically. After streaking, all the plates were incubated at 371˚C for 24h and the plates were observed for the growth of microorganism. The lowest concentration of the plant extract required for inhibiting the growth was considered as the MIC of the extracts against bacterial and fungal strains.

 

RESULTS:

Macroscopic characters:

The outer surface of the stem bark is pale grey and the inner surface is light brown. The stem bark exhibits deeply fissured surface forming thick vertically oblong hard chunks. Exfoliation through hard thick rectangular pieces; exposed surface is dark brown and the taste is bitter (Fig1).

 

Microscopic characters:

In transectional profile, the bark has deeper origin of periderm with wide irregular fissures. Periderm region is wider and comprises of narrow tabular phellem cells and equally developed inner phelloderm. Periderm is followed by very broad collapsed phloem. A distinct cambial zone is seen between the secondary xylem and secondary phloem (Fig2). Secondary phloem has regular radial files of sieve elements and fairly wide, straight phloem rays. The phloem elements are rectangular and thick walled comprising of sieve tube members, companion cells and tannin filled phloem parenchyma (Fig3). Collapsed phloem consists of thick tangential bands of sclerenchyma alternating with narrow bands of collapsed phloem. When viewed under polarized light microscope, the segments of phloem fibre are seen associated with prismatic calcium oxalate crystals (Fig4). In TLS view of phloem, the axial parenchyma exhibit dense vertical strands of prismatic crystals. These crystal strands are associated with phloem parenchyma. The crystals vary from cuboidal, rhomboidal and hexagonal morphological types. The phloem rays are mostly multiseriate, wide, high homocellular and non-storied (Fig5). In RLS view, the periderm exhibits wide zone of dark phellem and tannin free phelloderm. The secondary phloem shows wide horizontal bands of phloem rays. The phloem rays consist of horizontal layers of oblong homogenous cells with dense tannin contents. The vertical system shows phloem fibres, axial parenchyma with dense tannin accumulation (Fig6).

 

Powder microscopic observations

The bark powder is dark brown in colour with bitter taste and odourless. The microscopic study of powder revealed the presence of rhomboidal crystals of calcium oxalate, libriform fibres, thick masses of suberised phellem cells and tannin filled parenchyma cells.

 

Physico-chemical studies

The percentage of total ash, water-soluble ash, acid-insoluble ash, sulphated ash, alcohol- water soluble extractive value and loss on drying with reference to air dried powdered drug (Table 1).

 

Preliminary phytochemical screening

The preliminary phytochemical screening revealed the presence of alkaloids, phenolic compound, saponins and flavonoids in the EAPC extract while alkaloids, phenolic compound, saponins, flavonoids, phytosterol, proteins and amino acids in the HAPC extract (Table 2).

 

Antimicrobial activity

The results of the antimicrobial activities of both the extracts EAPC and HAPC from the stem bark of P. cineraria showed different degree of activity against the tested bacterial and fungal strains. The observed zones of inhibition and the MIC values of each extract against the tested bacterial and fungal strains were tabulated (Table 3 and 4).

 

Fig 6 RLS View

 

DISCUSSION:

Pharmacognostic study

In recent years there has been a rapid increase in the standardization of selected medicinal plants of potential therapeutic significance21,22. Despite the modern techniques, identification of plant drugs by pharmacognostic studies is more reliable. According to World Health Organization15 (WHO, 1998), the macroscopic and microscopic description of a medicinal plant is the first step towards establishing the identity and the degree of purity of such materials and should be carried out before any tests are undertaken. Macroscopically, the stem bark exhibits deeply fissured surface and rusty brown vertical patches. In transectional profile, deep seated first periderm and rhytidome type sequent periderm, dense tangential cylinders of xylem fibres, narrow, undilated, tanniniferous phloem rays and abundance of strand crystals of prismatic type associated with the xylem fibres are principal diagnostic characters of the bark. Further, the phloem rays are non-storied, multiseriate and homocellular. All these microscopic features can be employed to distinguish the bark samples of P. cineraria from its possible adulterants/substitutes. The aforesaid characters are stable and reliable features of the bark that are not influenced by environmental stress.

 

Fig 7a Antibacterial activity (Gram positive organisms)

 

Fig 7b Antibacterial (Gram negative organisms)

 

Fig 7c Antifungal activity

 

Fig 8a Antibacterial activity (Gram positive organisms)

 

Fig 8b Antibacterial activity (Gram negative organisms)

 

Fig 8c Antifungal activity

 

Table 4: MIC of stem bark of P. cineraria

S. No

Organisms

EAPC

HAPC

1.

Staphylococcus aureus

42

36

2.

Staphylococcus epidermidis

42

35

3.

Escherichia coli

41

39

4.

Klebsiella pneumoniae

44

40

5.

Aspergillus niger

44

41

6.

Aspergillus fumigatus

42

41

 

The physico-chemical evaluation of the drugs is an important parameter in detecting adulteration or improper handling of drugs. Ash value of a drug gives an idea of the earthy matter or the inorganic composition and other impurties present along with the drug. The ash values of the bark powder showed higher content of sulphated ash followed by total ash. Extractive values are primarily useful for the determination of exhausted or adulterated drugs. The water soluble extractive of the bark powder was high. The preliminary phytochemical screening of P. cineraria indicates the presence of various secondary plant metabolites in the extracts such as alkaloids, phenolic compound, saponins, flavonoids, phytosterol, proteins and amino acids that are known to possess various pharmacological effects and may be responsible for the various actions of P. cineraria.

 

Antimicrobial activity

Infectious diseases are a critical problem for health and they are the main cause of death worldwide. Resistance of microbes to antibiotics and toxicities produced by long term usage of antimicrobial compounds has initiated the search for safe antimicrobials23. Though different types of antimicrobial agents are available, there is an increase demand by people to use the natural products and also researchers have identified a lot of plants with antimicrobial activity24. Throughout the history of mankind, many infectious diseases have been treated with plant extracts. The different concentrations (50, 100, 150 g/disc) of HAPC and EAPC extracts of P. cineraria were tested against Gram positive, Gram negative bacterial strains and fungal strains. The higher concentrations of both extracts had inhibitory effects towards the tested microorganisms. HAPC of P. cineraria stem bark (Fig7a, 7b, 7c) was found to exhibit better inhibitory effects than EAPC (Fig8a, 8b, 8c) against S. aureus, S. epidermidis, E. coli and K. pneumonia but in case of A. niger and A. fumigatus, EAPC has shown better inhibitory effects and this effect was dose dependent. The extracts showed antimicrobial activity were subjected to minimum inhibitory concentration assay. In HAPC, the maximum inhibition against S. aureus (32mm) and for both S. epidermidis and E. coli (29mm) whereas in EAPC extract, the maximum inhibition against S. aureus (26mm) and S. epidermidis (29mm) at a concentration of 200 g/ml. The lowest MIC values were observed for HAPC (35 - 41 g/ml) and EAPC (41 - 44 g/ml) against the bacteria and fungi tested.

 

CONCLUSION:

The plant P. cineraria is useful in traditional medicine for the treatment of various ailments. So, it is important to standardize it for the purpose of therapeutic use. The pharmacognostic constants for the stem bark of this plant and the microscopic diagnostic features reported in this work could be useful for the compilation of a suitable monograph for its proper identification. The study of antimicrobial activity supports the traditional usage of the plant P. cineraria and suggests that the plant extracts possess compounds with antimicrobial and antifungal properties which can be used as antimicrobial agents in new drugs for the therapy of infectious diseases caused by pathogens. The most active extracts can be subjected to isolation of the therapeutic antimicrobials and carry out further pharmacological evaluation.

 

 

ACKNOWLEDGEMENT:

The authors would like to thank Prof. P. Jayaraman, Director of Plant Anatomy Research Centre for providing technical support during the studies.

 

 

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Received on 08.05.2009

Accepted on 14.08.2009

A&V Publication all right reserved

Research Journal of Pharmacognosy and Phytochemistry. 1(2): Sept. - Oct. 2009, 115-118