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The objectives of this study were to evaluate the antimicrobial activity and total antioxidant capacity (TAC) of licorice in Saliva of HIV/AIDS patients.
Saliva specimens were collected from 20 people living with HIV infection, with CD4 count <500 cells/mm3 from people infected with HIV/AIDS in Mangalore city, India. A combination of amoxicillin-clavulanic acid and nystatin was taken as the positive control and normal saline as negative control. Results were compared using one-way analysis of variance followed by Tukey's post hoc analysis in SPSS 19.
The TAC was evaluated spectrophotometrically at 695nm using the phosphomolybdenum method. Glycyrrhiza glabra showed a statistically significant reduction (P < 0.05) in total Candida count. The TAC of G. glabra was found to be 4.467 mM/L.
G. glabra extracts showed good anticandidal activity and also high antioxidant property which reduces the oxidative stress of HIV-infected people.
HIV/AIDS has emerged as a major public health problem since its recognition as an emerging disease a couple of decades ago. According to the estimates of the National AIDS Control Organization, India had 2.2 million HIV-positive persons in 2008, with an estimated HIV prevalence of 0.29% among adults, and Karnataka is one among the high prevalence states of South India. HIV infection induces a wide array of immunologic alterations resulting in the progressive development of opportunistic infections and malignancy, which results in AIDS. Due to widespread emergence of resistance among microbial pathogens against antibiotics, there is an immense requirement to discover novel antibiotics. Traditional medical practitioners use different medicinal plants for curing various diseases in their daily practice, but a rational approach of this with the modern system of medicine is still not available. HIV-infected people and AIDS patients often seek complementary therapies including herbal medicines due to reasons such as unsatisfactory effects, high cost, nonavailability, or adverse effects of conventional medicines.
Glycyrrhiza glabra L., family Fabaceae, known as licorice, is a plant growth belonging to the pea and bean family, which has extensive use in foods and both traditional and herbal medicine. It is a herb which is cultivated mainly for its underground stems that are used to flavor confectionery. It is also known as “sweet roots,” which contains a compound that is roughly 50 times sweeter than sugar.[6,7] Recent research has indicated that it can slow down the progression of HIV to AIDS, by triggering the chemical compound interferon.
Infection with viruses such as HIV causes persistent chronic inflammation. It's protein produces a progressive intracellular increase of the reactive oxygen species, which influences the rate of apoptosis, decreases CD4 lymphocyte cells, and consequently increases HIV replication secondary to over production of free radicals. This actually interferes with normal physiological chemistry of the endoplasmic reticulum and cell mitochondria. Such interference is responsible for the generation of oxygen reactive species and depletion of total antioxidant capacity (TAC) in AIDS patient.
The antioxidant and antimicrobial constituents of licorice were studied by some researchers,[5,6,9,10,11] but there is a dearth of knowledge regarding their effectiveness while comparing with the routine gold standard antibiotics. Based on ethnobotanical knowledge, an attempt was made here to assess the TAC and antimicrobial activity of G. glabra in saliva of people living with HIV/AIDS.
The present study was a cross-sectional study conducted at Snehasadan, which is a care and support center for people infected with HIV/AIDS situated in Mangalore, India. Ten HIV-infected patients were enrolled in the study, who readily participated. Ethical approval was taken from the Institutional ethical committee as well as the Social Welfare Department, Mangalore city. HIV patients with no acute opportunistic infection and who gave informed consent were included for the study. Patients with hyperlipidemia, diabetes, kidney or liver dysfunction, intractable diarrhea (more than six liquid stools per day), vomiting, or evidence of gastrointestinal bleeding were excluded from the study.
The dried roots of licorice were powdered in a mixer grinder and were then packed in paper bag and stored in airtight containers until use. Powered material was extracted with chloroform, acetone, and ether by soxhalation method. Extracts were evaporated in hot air oven at 45°C over night. The aqueous extract was then used for further antimicrobial assay.
A combination of amoxicillin, clavulanic acid, and nystatin was taken as the positive control, and normal saline was taken as the negative control.
Saliva samples were collected from subjects who had a CD4 count <300 cells/mm3 of blood, in a sterile container under aseptic precautions. Subjects were asked to chew the wax block to stimulate salivation and collect the saliva for a minute. Stimulated saliva was sucked from the mouth of subjects and was injected into containers containing thioglycollate broth with hemin and vitamin K transport media. After that, it is stored in an ice box at 4°C and transported to Research Laboratory, NITTE University, Deralakatte for analysis.
MacConkeys agar plates were used to isolate Klebseilla species, sabouraud dextrose agar plates for Candida species, and sheep blood agar plates for alpha hemolytic streptococci. The plates were incubated at 37°C for 2 days. Bacterial identification was done using colony morphology, gram staining, and biochemical tests. Colony counts were determined using colony counter. Numbers of colony forming units were multiplied by the dilution factor to obtain the number of colony forming units/4 cm2. TAC was assessed spectrophotometrically at 695 nm using the phosphomolybdenum method.
The results were analyzed using SPSS version 19. One-way analysis of variance (ANOVA) followed by Tukey's post hoc test were used to compare the difference in the colony forming units. A difference was considered to be of statistical significance if the P < 0.05
A total of 20 volunteers participated in the study out of which 60% were females and 40% were males. The mean age of participants was 42.7 years.
Table 1 shows the comparison of G. glabra, normal saline (negative control) and amoxicillin-clavulanic acid and nystatin in combination (positive control) in reducing the candida, Klebsiella and α-hemolytic streptococcal counts by one-way ANOVA followed by Tukey's test. G. glabra showed a statistically significant reduction (P = 0.025) in total Candida count. No significant reduction is seen with total Klebsiella and α-hemolytic streptococcal counts (P > 0.05).
When comparing the candida colony forming units by, it was found that the mean Candida count with G. glabra (7.63 × 102) showed a significant difference with the negative control (84.62 × 102)(P < 0.05) whereas mean candida count did not show much difference with the positive control of antibiotic combination (6.42 × 102)(P < 0.12) [Graph 1]. The TAC of G. glabra was estimated to be 4.467 mM/L by the phosphomolybdenum method.
The world health organization estimates that about 80% of people living in developing countries rely mostly on traditional medicines for their primary health needs. Herbal preparations are cheap and simple; hence, they remain a hope for the HIV/AIDS-infected people with opportunistic infections.
In the present study, G. glabra was found to possess good anticandidal activity. The anticandidal activity and antimicrobial properties exhibited by the licorice were shown in studies conducted by Meghashri and Gopal and Janovska et al. Even though there is a small reduction in Klebsiella and α-hemolytic streptococcal counts, it was all surpassed by the significant anticandidal activity. In one study by Vivek et al., it was shown that gabardine, one of the most important substances in licorice, had antibacterial activities against some strains, and it was more active against Gram-positive strains than Gram-negative. Polysaccharides from G. glabra also enriched with glucuronic acid, showed strong antiadhesive properties against various pathogens. Licorice extracts have been widely used in pharmaceutical and confectionery industries because of the presence of glycyrrhizin. Glycyrrhizic acid or glycyrrhizin is the main biologically active compound of the liquorice root.
Saliva can constitute a first line of defense against free radical-mediated oxidative stress. The use of saliva as a diagnostic fluid has become somewhat of a translational research success story. In the present study, the TAC of G. glabra was estimated to be 4.467 mM/L. Similar studies were conducted by various researchers.[8,18] It has been observed that perturbations in the antioxidant defense systems, and consequently redox imbalance are present in many tissues of HIV-infected patients. Hence, the exogenous supply of antioxidants, as natural compounds that scavenge free radicals, might represent an important additional strategy for the treatment of HIV infection. Such screening of various natural agents becomes successful in the prediction of lead molecule and drug-like properties at the onset of drug discovery will pay off later in drug development.
The results from this study indicate that G. glabra extracts showed good anticandidal activity. It also got a high- antioxidant property which reduces the oxidative stress of HIV-infected people and thus can be used to treat many diseases including bacterial and fungal infections. The potential use of this natural product as an adjuvant treatment in patients infected with HIV/AIDS needs to be further explored.
There are no conflicts of interest.