DOI: http://dx.doi.org/10.26510/2394-0859.pbe.2017.37

Research Article

Antimicrobial evaluation of flowering stalks of Cistanche violacea, a holoparasitic plant collected from arid region in Qassim, Saudi Arabia

Emad Mohamed Abdallah*

Department of Laboratory Sciences, College of Sciences and Arts, Qassim University, Al-Rass, Saudi Arabia

*For correspondence

Dr. Emad Mohamed Abdallah

Department of Laboratory Sciences, College of Sciences and Arts, Qassim University, Al-Rass, Saudi Arabia.

Email: 140208@qu.edu.sa

 

 

 

 

 

 

 

 

Received: 22 October 2017

Accepted: 19 November 2017

ABSTRACT

Objective: Cistanche violacea has been found in Qassim region (Saudi Arabia), growing as a parasitic plant on Haloxylon salicornicum. The flowering stalk part of this plant was evaluated for antimicrobial potential against various referenced bacteria and fungi.

Methods: The flowering stalk parts were collected from the field, dried, extracted by maceration in 80% methanol and 80% Ethanol, The crude extracts were examined against the tested microorganisms using agar-well diffusion method.

Results: The antimicrobial testing showed that among 10 referenced bacterial isolates, only one exhibited significant susceptibility to the methanol and ethanol extracts of C. violacea, which was Staphylococcus epidermidis ATCC® 12228TM, recorded 15 and 16 mm inhibition zones, respectively. While other bacteria recorded weak or no susceptibility and no antifungal activity was detected.

Conclusions: In general, the methanolic and ethanolic extracts of the flowering stalks of C. violacea exhibited weak or no antimicrobial properties except with one bacterial strain, further studies on the other biological activities are recommended as well as investigating the chemical constituents. To the best of the author's knowledge, this is the first report evaluating the antimicrobial potential of the flowering stalks of C. violacea in Qassim area, Saudi Arabia.

Keywords: Antimicrobial, Antibacterial, Antifungal, Agar-well diffusion, Cistanche violacea, Holoparsitic plant

Introduction

Exploration of the healing power of natural products is an ancient and established belief. However, the current civilization, which initially depends on the industrial revolution, has developed the organic chemistry and introduced it in pharmacology, resulted in a preference for synthetic drugs in modern medicine over natural products.1 On the other side, medicinal plants still retain its popularity, it was estimated that up to 80% of the world inhabitants depends on plants to cover their primary healthcare needs.2 It is thought that there are 250 000 to 500 000 known species of plants on earth, these plants are considered as a large diverse resource for biological and chemical products of bioactive effects on the human body.3 Surprisingly, there are 25 to 50% of current drugs prescribed in modern medicine are derived from plants; none of them is used as antimicrobial drug.4 Nowadays, the lack of effective antimicrobial drugs are a major health problem. This problem has led to growing interest in evaluating the antimicrobial activity of plants all over the globe and numerous studies have been published, some of them showed potent antimicrobial effects even against multidrug-resistant microorganisms, but unfortunately little of them have attracted the attention the pharmaceutical companies in order to be used in the near future as a drug.5 The desert Hyacinth or Cistanche violacea (Desf.) Beck. in Engl. and Prantl. is belonging to family Orobanchaceae. It is the largest parasitic family in the plant kingdom, which could be either facultative parasites (Photosynthetic/ hemiparasitic) or totally-dependent parasites (Holoparasitic), dominated in the dry tropics or subtropics regions.6 There are about 22 known species of Cistanche in the world; these perennial holoparasitic plants lack chlorophyll, so it absorbs water and nutrients from the roots of the host plant.7 In fact, the flora of the Arabian desert has been extensively studied. However, little is known about Cistanche species and other angiosperm parasites and received no attention as it is found in low percentage among other desert vegetation in the arid zones of the Middle East and North Africa.8 Accordingly, information about the bioactivity of C. violacea is very scant. However, some species are used in traditional medicine; for example; C. deserticola is widely used in Chinese herbal medicine, its main chemical compound is phenylethanoid, which claimed to possess hepatoprotective, anti-inflammatory, antinociceptive, antioxidant, sedative and improves sexual activity.9 The aim of this study is to evaluate first the antimicrobial potential of the areal parts (flowering stalks) of C. violacea, later studies will continue on the other parts of this neglected parasitic plant.

Materials and Methods

Collection of plant material

The flowering stalk parts of Cistanche violacea were collected from the arid areas in Qassim region, Saudi Arabia, during the short springtime in February 2017. In the collection area, the plant was authenticated by Prof. Dr. Gamal E. El-Ghazali, a distinguished taxonomist from College of Sciences and Arts, Qassim University. Cistanche violacea was found parasitizing a desert plant known as Haloxylon salicornicum (Figure 1). Only flowering stalks are appeared in the spring, while all other parts (Rhizome) are located underground. The flowering stalks were collected in the morning and directly transported to the laboratory, cut into small parts with scissors and kept in the oven at 50oC for up to a week until dried. The adjustment at 50oC is required to avoid any fungal growth on the moisten plant material.

Figure 1: The flowering stalks of the parasitic plant C. violacea.*

*A= Parasiting on haloxylon salicornicum;

B= flowering stalks several meters far from its host.

Plant extraction

The dried flowering stalks of C. violacea was ground with a grinder to a fine powder. 50 grams of that powder was weight and macerated in 80% methanol, another 50 grams of the plant' powder was weight and macerated in 80% ethanol. Each macerate was kept in a well-tighten dark glass container and kept for up to 3 days inside the incubator at 37oC. Thereafter, the macerate solution was filtered using a muslin cloth, followed by filtration with Whatman filter paper No.1. The methanol and ethanol extracts were evaporated under reduced pressure using rotary evaporator to get semisolid crude and it was directly put in an incubator at 50 oC two days to get dry crude, which kept the fridge in a dry closed container until used.

Tested microorganisms

Microorganisms used in this study to evaluate the antimicrobial activity of the flowering stalks of C. violacea were American type culture collection (ATCC), which are enlisted in (Table 1).

Antimicrobial test

The antimicrobial activity of the methanol and ethanol crude extracts of C. violacea was evaluated using agar-well diffusion method as reported by (Mekonnen et al) with minor modifications.10 20 ml of autoclaved Mueller-Hinton agar for bacteria or sabouraud dextrose agar for fungi was poured on sterile disposable plastic peri-dishes (90 mm in diameter) and left to solidify at room temperature. Then, 16–24-hour-old cultures of bacterial strains or 48-hour-old cultures of fungal strains were spread over the agar plates using a sterile cotton swab. 3 wells for each bacterial plate or one well in the center of each fungal plate were punched in the agar surface using sterile/flamed cork borer (6 mm in diameter). 500 mg of the dry crude of methanol or ethanol extract was reconstituted in 1 ml of 10% DMSO (Di-methyl sulphoxide) to make 500 mg/ml. 100 µl from that concentration was pipette and loaded into respective wells, also 100 µl of chloramphenicol (5 mg/ml) for bacteria or clotrimazole (10 mg/ml) for fungi was loaded to a separate well and served as positive control. The pre-experimental testing showed that 10% DMSO has no effect on bacterial or fungal growth. The seeded plates were incubated for 24 hours at 37oC for bacteria or for 48 hours at 25-28oC for fungi. After incubation, the clear zones of inhibition were measured using a ruler and the results recorded in millimeters (mm).

Table 1: Microorganisms used in the evaluation of antimicrobial properties of flowering stalks of C. violacea.

Type of microorganisms Scientific name Strain code
Gram-positive Bacteria Bacillus cereus ATCC® 10876TM
Staphylococcus epidermidis ATCC® 12228TM
Staphylococcus aureus ATCC® 29213TM
Staphylococcus saprophyticus ATCC® 43867TM
Streptococcus pneumonia ATCC® 49619TM.
Gram-negative bacteria Escherichia coli ATCC® 25922TM
Proteus vulgaris ATCC® 6380TM
Klebsiella pneumonia ATCC® 27736TM
Pseudomonas aeruginosa ATCC® 9027TM
Shigella flexsneri ATCC® 12022TM
Fungus Aspergilllus niger ATCC®6275TM
Yeast Candida albicans ATCC®10231TM

Results and Discussion

A hydro-alcoholic extracts (80% methanol and 80% ethanol) of flowering stalks of C. violacea were tested against a wide variety of microorganisms (Table 1), in order to evaluate its potential antimicrobial properties. The results revealed that only one microorganism exhibited significant susceptibility to the methanol and ethanol extracts of C. violacea, which was Staphylococcus epidermidis ATCC® 12228TM, recorded 15 and 16 mm inhibition zones, respectively. While other microorganisms recorded weak or no susceptibility and no antifungal activity was detected (Tables 2, 3 and 4), these results become more clearer when omitting the diameter of the well, which is 6 mm in size, as represented in Figures (4, 5 and 6). In addition, some representative photos are added to show the presence or the absence of the zones of inhibition on the plates (Figure 2 and 3).

Table 2: Antibacterial properties of flowering stalks of Cistanche violacea against gram-positive bacteria.

Tested Zone of Inhibition (mm)
Bc Se Ss Sa Sp
Methanol extract (500 mg/ml) 7 15 6 7 6
Ethanol extract (500 mg/ml) 8 16 6 8 6
Chloramphenicol (5 mg/ml) 32 38 31 34 33

Bc=Bacillus cereus ATCC® 10876TM, Se=Staphylococcus epidermidis ATCC® 12228TM, Ss=Staphylococcus saprophyticus ATCC® 43867TM, Sa=Staphylococcus aureus ATCC® 29213TM, and Sp=Streptococcus pneumonia ATCC® 49619TM. Zone diameter equals 6 mm= no inhibition.

Table 3: Antibacterial properties of flowering stalks of Cistanche violacea against gram-negative bacteria.

Tested Zone of Inhibition (mm)
Ec Pv Kp Pa Sf
Methanol extract (500 mg/ml) 6 7 6 7 6
Ethanol extract (500 mg/ml) 6 7 6 8 6
Chloramphenicol (5 mg/ml) 36 33 32 16 31

Ec=Escherichia coli ATCC® 25922TM, Pv=Proteus vulgaris ATCC® 6380TM, Kp=Klebsiella pneumonia ATCC® 27736TM, Pa=Pseudomonas aeruginosa ATCC® 9027TM and Sf=Shigella flexsneri ATCC® 12022TM. Zone diameter equal 6 mm= no inhibition.

Table 4: Antifungal properties of flowering stalks of Cistanche violacea.

Tested compound Mean zone of inhibition (mm)
Aspergillus niger Candida albicans
Methanol extract (500 mg/ml) 6 6
Ethanol extract (500 mg/ml) 6 6
Clotrimazole (10 mg/ml) 22 26

In details, S. epidermidis recorded 15 and 16 mm inhibition zones with Methanol and ethanol respectively. This result considered a good antibacterial activity. The previous Study considers the inhibition zone from 14 mm and above, as high antimicrobial activity.11 S. epidermidis is a member of the normal microflora on the human body, colonizes predominantly some parts of the skin but under some circumstances, it becomes an opportunistic pathogen.12 With reference to the results, the extracts of the flowering stalks of C. violacea showed some degrees of antibacterial activities but all were less than 14 mm zone of inhibition and considered as weak activity, these bacteria were; B. cereus (8 and 7 mm), S. aureus (8 and 7 mm), P. aeruginosa (8 and 7 mm) and P. vulgaris (7 and 7 mm) for ethanolic and methanolic extracts, respectively. While all other bacteria was not susceptible to any extract, these bacteria are S. saprophyticus, S. pneumonia, E. coli, K. pneumonia and S. flexsneri. In addition, A. niger and C. albicans showed no susceptibility towards the methanolic and the ethanolic extracts of the flowering stalks of C. violacea. The current study is compatible with the previous study on C. phelypaea from Sudan, where it reports that the methanolic and aqueous extracts of C. phelypaea areal parts have weak or no antibacterial activity.13 Moreover, the areal parts of C. tubulosa exhibited mild antifungal and antibacterial effects against different microorganisms.14 No doubt that, the noticeable antibacterial activity of the flowering stalks of C. violacea against S. epidermidis in the current study is attributed to some phytochemical compounds present in this parasitic plant may be these active compounds are in low quantities in the flowering stalks or the nature of the compounds are more hydrophobic and non-polar, hence study on the essential oils in this parasitic plants is recommended. The other prospective assumption is that the bioactive compounds are found in the rhizome underground and not in the superficial parts of the ground. In one of the rare studies on C. violacea in Tunisia, the phytochemical investigation revealed high phenolic contents in the flowers and high flavonoids contents in the bulb of C. violacea.15 Moreover, phytochemical studies on some Cistanche species reported the presence of some important compounds such as polysaccharides, oligosaccharides, glycosides, phenylethanoid glycosides, coumarins, resin and tannins, iridoids, lignans, alditols and flavonoids.7 To the best of my knowledge, and after an intensive search in literature and sources of information, this study is the first report evaluating the antimicrobial potential of the flowering stalks of C. violacea in Qassim area, Saudi Arabia. The report of (Debouba et al) supports my claim, who stated that "No available data on C. violacea could be found in the literature".15 In addition, Fahmy cited that, the angiosperm parasites are neglected in the studies and investigations on the flora of the Arabian deserts in the Middle East and North Africa.8

Figure 2: Representative photo showing susceptibility of some bacterial strains to C. violacea extracts compared with antibiotic.

Figure 3: Representative photo showing susceptibility of fungal strains to C. violacea extracts.

Figure 4: Mean zone of inhibition gram-positive bacteria, extracts and antibiotic.

Figure 5: Mean zone of inhibition gram-negative bacteria, extracts and antibiotic.

Figure 6: Mean zone of inhibition fungi, extracts and antibiotic.

Conclusions

In the current study, methanol and ethanol extracts of the flowering stalks of Cistanche violacea exhibited weak or no antimicrobial activity except good activity with one gram-positive bacterium (Staphylococcus epidermidis), which generated the assumption that either the antibacterial compounds are present in low quantities or its chemical nature require extraction with non-polar or hydrophobic solvents. More studies are required in order to determine its phytochemical properties as well as other bioactive potential. It is also recommended to investigate the bioactive and antimicrobial properties of the perennial rhizome, which found deep in the soil, attached to the root of the host plant.

Acknowledgements

The author would like to thank Prof. Dr. Gamal Al-Ghazali for authenticating the tested plant and providing valuable botanical information about it. Thanks also to Dr. Adel Abbas, department of chemistry, College of Sciences and Arts in Rass, for his generous help in evaporating the extracts with Rotary Evaporator.

Funding: No funding sources

Conflict of interest: None declared

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