Background
Since thousands of years herbal products have been the basis for medical treatments and are recognized as a form of alternative medicine. Modern medicine also employs use of many herbal products for therapeutic purposes [
1]. The emerging trends of various antimicrobial compounds is alarming; and from last few years researchers are in attempt to search new alternative medicines for treatment of different infectious diseases [
2]. Herbal medicines are specially recommended for those patients who cannot use chemical products due to their adverse and biochemical contraindications [
3,
4].
World Health Organization (WHO) estimates that 80% of the population in developing countries employs the usage of herbal medicine to treat aspects of primary health care. These medicines also serve as precursor for 20 to 25% of modern medicine as these herbal medicines are considered to be economical, safer and less toxic [
5,
6].
Three medically acclaimed plants were included in this study:
Miswak (
Salvadora persica) a medicinal herb which is commonly available and very cost effective. It is reported to have antibacterial, antifungal, antioxidant and anticancer properties [
7,
8].
Aloe vera is a medicinal herb whose mucilaginous gel is traditionally used to treat different diseases. It is very popular among scientists for its medicinal properties [
9]. Of the overall weight of
Aloe vera 2% consist of the active compounds which include methylchromones, flavonoids, aloesin, aloe-emodin, sterols, amino acids, aloemannan, aloin, acemannan, aloeride, naftoquinones, saponin and vitamins. It is considered to be antibacterial, anti-inflammatory and antioxidant [
10,
11]. Another important medicinal herb
Nigella sativa (
Kalonji) used in this study is reported to possess antimicrobial, anti-inflammatory, antiulcer, anticancer, immune stimulating and antioxidant activity [
12‐
14]. Phytochemical analysis of
Nigella seeds revealed that it contains a variety of volatile oil and fixed oil and other components including Nigellin, Carvone, Melanthin, Carvene, Cymene and Thymoquinone [
14]. A lot of medicinal plants elaborate variety of compounds majority of which have extremely important properties particularly antimicrobial activity and some herbs also possess anticancer activity. Many medicinal herbs are reported to cure different acute and chronic infections including dental infections. Dzoyem in 2016 also have reported antimicrobial activities and anti-cancer activities of fourteen herb against common pathogens [
15].
Herbs to herb combinations have been traditionally used in different regions around the word form thousands of years, however scientific evidence based data is still lacking. Traditionally co-administration of herb is believed to influence the overall effect of herb, either complementary or antagonistic [
16]. Herbs are believed to contain several potential sources of potent biological compounds. Using herb in combination with others herb or along with antibiotics can produce additional benefits and also reduce the toxicity of herb. Generally herbal combinations are used due to number of reasons as it is believed that their use in combination may give rise to synergistic or additive effect which ultimately helps to overcome drug resistance and increase the spectrum of activity. It may reduce the required dose of administration of any drug and reduce the overall cost along with the side effects [
17]. A similar concept in modern medicine is the use of “cocktail” in antiretroviral therapy (HAART) [
16]. However less data is present to use these herbs in combinations against oral pathogens. So this study was aimed to ascertain following objectives; to determine antimicrobial activity of these herbs (
Kalonji, Miswak and Aloe Vera) against oral pathogens, to evaluate anti-proliferative activity against
HeLa cell line and also antioxidative ability.
Discussion
The results of this study indicate that medicinal herbs
Miswak, Kalonji &
Aloe vera contain significant level of antimicrobial, antioxidant and anti-proliferative agents in them and hence needed to be further explored. Production of synthetic drugs effectively improves the health care facilities around the world. In developing countries still local herbal products are common to treat a lot of diseased conditions due to their cost effectiveness and less side effects [
22]. Fabricant & Farnsworth in 2001 reported active compounds obtained from herbal products have 80% correlation between their traditional use and use in modern therapeutics [
16,
23]. Rath S and Padhy RN in 2015 stated that therapeutic plants have immense potential to be used in integrative and synergistic drug development [
24].
Test organisms in this study
Staphylococcus aureus and
Candida albicans are the most common cause of oral infections across the world [
25,
26]. Our findings of herbal extracts affectivity against oral pathogens are in consistence with previous reports [
7,
8,
27]. Al-Obaida and coauthors declared
miswak as an effective oral hygienic tool and found it helpful against different organisms causing oral cavity infections [
28,
29]. A study conducted by Heshama A.E 2016 stated that the beneficial effects of
miswak on oral health are due to presence of different important minerals and compounds. The combination of Benzyllisothiocyanate, salvadorine, fluoride, tannins, vitamin C, Silica, chloride and essential oils contribute to the value of
miswak [
30].
The results of this study are comparable with the previous studies as the prepared (PM)
Miswak extract produced significant zone of inhibition against
Candida albicans but contrary to this aqueous and ethanol extracts were found ineffective when applied on both test organisms. Another study showed that aqueous and ethanolic extracts of
Miswak produced significant zone of inhibition against
Staphylococcus aureus and
Candida albicans [
27]. Salehi et al. also showed that
Miswak extracts used in form of mouthwash were effective against oral pathogens [
29]. Another study reported reduction w?>in number of organism by the use of
Miswak extracts as mouth wash [
27]. Omer et al. stated that
Miswak extracts when prepared in solvents chloroform, ethyl acetate and ethanol it produced no zone of inhibition against
Candida albicans and
Staphylococcus aureus but its aqueous extract was effective [
31]. In contrast to Al-Obaida et al. reported herbal ethanol extracts were effective against
S. aureus and C. albicans. In 2013 Bafti et al. reported
Miswak extracts contain good antifungal properties [
3]. Finding of our study also suggest
Miswak as an antioxidant agent it contain antioxidant compounds for example peroxidase, catalase and polyphenol oxidase representing it as a good chewing stick [
7].
These inconsistent findings of
miswak extract are might be due to the difference in the environmental, climate, and harvesting conditions of herbs. The results of our study show some difference as compared to the published data. But there are inconsistent reports regarding herbal activities. These variation in the activity of herb results are comparable with other studies including Teka et al. [
32], Darout et al. [
22], Bakathir and Abbas [
33], Togan et al. [
18], Khan et al. [
14]. Findings of these authors suggest that variation in results of herbs when applied against organism can come due to many reasons for example geographical location, harvesting conditions, chemical constituents and use of different techniques of extraction. Activity of the herb also varies if the organism is isolated from clinical samples or standard strains [
18,
22,
32‐
34].
Aloe vera being a phytotherapic agent also act as inhibitory factor for the growth of wide range of oral microorganisms and is reported to significantly reduce intensity of gingivitis and plaque formation [
35,
36]. However, in this study
Aloe vera extracts did not exhibit antimicrobial activity in consistent with previous studies where it’s aqueous and methanol extracts were found effective against
Candida albicans but were ineffective against
Staphylococcys aureus [
37].
Kalonji also called miracle herb due to its medicinal properties. It is a commonly grown herb all over the world. In herbal medicine its seeds are commonly used to treat many diseases [
14,
38]
. Many studies reported it’s antioxidant, antibacterial, antifungal, anti-plaque and anti-proliferation activities [
14]. The results of our study also showed similar findings as extracts prepared from
Kalonji showed significant antioxidant and anti-proliferative abilities especially when extracted in ethanol and petroleum ether but not antimicrobial activity. A study conducted by Khan et al. 2013 in Pakistan also show comparable result to our finding where
Nigella extracted in methanol was found ineffective when applied against
S. aureus. Studies also suggested that biologically active medicinal plants can show inhibitory activity against some organism but can be ineffective against others [
34].
Our finding that extracts in combinations produce significant antimicrobial activity, when applied on both test organisms
(P <0.001
) is striking. In some extracts when they were tested as alone extracts were not active against
Staphylococcus aureus while when tested in combinations with other extracts significant zone of inhibitions were produced this may be attributed by the synergistic and antagonistic effect produced by constituents of herbal extract [
39,
40].
The study conducted by Bag et al. in 2015 also enforce that when two herbs are given in combination the biological effects of herbal combination is enhanced and show synergistic effect [
17]. This synergistic effects are due to the interrelationships of herbal ingredients where synergy can be broadly classified into two main categories based on the mode of actions including pharmacodynamic (when two or more constituents work on same biological targets and result in improved therapeutic outcomes) and pharmacokinetic (two or more constituents interactions between their metabolic processes, absorption, distribution and elimination) [
41].
This study was designed with the hypothesis that medical herbs contain potent antitumor and anti cancerous agent. We examined the effect of
Kalonji, Miswak and
Aloe vera extracts in petroleum ether, ethanol and water at a concentration of 1 mg/ml. Findings suggest only
Kalonji extracted in petroleum ether and in ethanol have anti-proliferation activity with a statistical border line significant
p value. These results are in agreement with previous studies already reported anti proliferative and antioxidant activity of herbs [
20,
27,
29].
As the effectiveness of these herbs in combination have not been previously explore that provides this study more strength. However, there are few limitations of this study; due to limited availability of extract it was not possible to test herbal combinations at different concentrations and it was also not possible to make further herbal combinations so only 18 different combinations of extracts were used. This small volume of our extracts also made it impossible to test anti-proliferative activity on other cell lines this leads to uncertainty in these results. However it is recommended to further check the activities of these herbs on other cell lines.
This study suggests medicinal herbs particularly
Miswak and
Kalonji have potential to be used for therapeutic purpose. These medicinal herbs should be tested in further combinations to determine their medicinal potentials.
Miswak’s therapeutic efficacy can be increased in many folds in combination with other medicinal herbs hence it should be further investigated. More research work should be conducted in the field of phytochemistry focusing on both in vitro and in vivo analyses and the isolation of bioactive compounds of interest [
42].
Acknowledgements
We are thankful to Mr. Muhamad Shoaib (Herbalist) to identify our herb samples used in this study.