This cross-sectional comparative study was conducted on 40 male subjects in each group over years (March 2015- April 2016) that were randomly selected by convenient sampling technique. Forty healthy non-smoker males formed the control group (Group A). Age, height and weight matched [Table/Fig-1] male chronic smokers indulging in heavy tobacco smoking mostly in the form of cigarettes 80 to 100 per week for the last 8-10 years formed the experimental group (Group B). The subjects of both groups recruited were from the hospital workers and attendants of the patients having lower socioeconomic status. Both the hospital workers and attendants of the patients were lower grade office staffs by occupation with almost sedentary lifestyle having no physical exertion.

The subjects with acute illness or any respiratory illness at the time of the study were excluded. Subjects were also excluded if satisfactory measurements could not be taken because of lack of cooperation or an inability to perform the tests. Prior to the study, all subjects were informed of the study procedure, purpose and known risks and thereby obtained their informed written consent. This study was approved by the Institutional Ethical Committee, Chitwan Medical College, Nepal.

All the subjects of Group A and Group B were subjected to pulmonary function tests- FVC, FEV₁, FEV₁/FVC ratio and PEFR in sitting upright posture. Computerized equipment “MEDSPIROR” (MED SYSTEMS Pvt. Ltd., Chandigarh, India) was used in this study. Standard methods and precautions outlined by Miller M et al., have been followed. Pulmonary function tests were carried out at a fix time of the day (10.00 – 12.00 noon) to minimize any diurnal variation [15]. All values were corrected for body temperature and pressure saturated with water vapour.

For this study, some food items (vegetables, fruits and spices) were screened for the production of nitric oxide in vitro which has been described below in detail in the headings with “Preparation of extract of various vegetables, fruits and spices” and “Assay of nitric oxide synthase activity of the extract”. Among them, garlic has shown the highest NOS stimulating activity [Table/Fig-2]. In this experimental design, the subjects of Group B were then supplemented with approximately 4 gm (2 cloves) of raw garlic per day for three months, garlic dose recommended in the study by Tattelman E [16] and the University of Maryland Medical Center [17], referred as experimental group II (Group C). At the end of three months of garlic supplementation the subjects of Group C were again subjected to pulmonary function tests. Endogenous nitric oxide production was studied in smokers before (Group B) and after (Group C) garlic supplementation. Endogenous nitric oxide production was also studied in non-smokers (Group A) at the start of the study.

Blood was collected in plastic tubes containing 0.13 M sodium citrate. Intact red blood cells were isolated and suspended in 0.9% NaCl; RBC membranes were prepared by ultra-centrifugation of red blood cell lysate for the measurement of nitric oxide level by activation of nitric oxide synthase enzyme present in RBC cell membrane [18]. For estimation of endogenous production of nitric oxide, the blood was collected before (Group A and Group B) and after (Group C) garlic supplementation and the RBC was processed as described above for in vitro production of nitric oxide.

Various vegetables, fruits and spices were homogenized in glass Potter Elvejyeum homogenizer by suspending in Kreb’s buffer, pH 7.4 (5 mg/ ml) at 0°C. Some vegetables, fruits and spices were homogenized after boiling in test tube with a tight cork. The homogenized mass was then centrifuged at 60,000 g at 0°C for 60 minutes in separate tubes. Supernatant hereafter called as “extract” was collected and protein concentration was determined according to Lowry OH et al., [19].

Different extracts were separately incubated at 37°C with constant stirring for 30-45 minutes with Kreb’s buffer. The incubation mixture was centrifuged at 0°C at 8,000 gm for 20 minutes and supernatant was collected for determination of nitric oxide synthase, which was expressed as nmol NO/ mg protein/hr [20].

Nitric oxide was determined according to Jia L et al., by using Scanning Beckman Spectrophotometer (Model DU6, USA) [21]. Nitric oxide content of the supernatant was quantitated by determining the conversion of oxyhaemoglobin to methaemoglobin.

All values were expressed as mean±SE, Unpaired student’s t-test was used for groupwise comparisons and p-value of <0.05 was considered statistically significant.

Some vegetable, fruits and spices were screened for the production of nitric oxide in vitro. The nitric oxide production ranged from 0.00 to 20.08 (nmol NO/mg protein/hr). Those items produced more nitric oxide is presented in [Table/Fig-2]. Among them, garlic has shown the highest (20.08 nmol NO/mg protein/hr) nitric oxide production capability.

Nitric oxide synthase activity showed a significant (p<0.01) decrease in smokers (Group B) compared to non-smokers (Group A) [Table/Fig-3]. Garlic supplementation had significantly (p<0.05) increased the nitric oxide synthase activity of the smokers (Group C).

[Table/Fig-4,5] show that forced vital capacity was markedly reduced (22%) in smokers (Group B) as compared to non-smokers (Group A) which was statistically significant (p<0.01). Supplementation of garlic significantly (p<0.05) improved the forced vital capacity of the smokers (Group C).

As for FVC, the FEV₁ was also significantly (p<0.01) reduced by 16% in smokers as compared to non-smokers [Table/Fig-4,5]. Dietary supplementation of garlic has significantly (p<0.01) improved (13%) the FEV₁ in smokers (Group C) as compared to Group B.

PEFR of Group B was significantly (p<0.01) reduced by 17% as compared to Group A [Table/Fig-4,5]. After supplementation of garlic, PEFR was improved significantly (p<0.05) in Group C compared to Group B.

[Table/Fig-4,5] shows that FEV₁/VC ratio was marked reduced (16.3%) in smokers (Group B) as compared to non-smokers (Group A) which was statistically significant (p<0.05). Supplementation of garlic significantly (p< 0.05) improved the FEV₁/FVC ratio of smokers (Group C).

Our findings showed that FVC, FEV₁, FEV₁/FVC ratio and PEFR were decreased significantly in smokers compared to non-smokers [Table/Fig-4,5]. Though, these reductions are quite significant statistically in all these parameters in smokers, they were clinically asymptomatic during the study, which might be due to their sedentary lifestyle and concurrent effect of chronic smoking. These findings are well supported by findings by Bohadana A et al., and Baraldo S et al., with explanation to the possible mechanism of these reductions of pulmonary function test in tobacco smoking due to narrowing of small airways [1,2]. This causes reduced elastic recoil, increased compliance and increased physiological dead space [1]. These smokers with reduced PFT parameters did not visit to any clinicians as they did not have clinically apparent difficulties in respiration with daily normal life activities. This interpretation finds support from earlier reports that this might be due to undiagnosed respiratory problems, as in UK, it is estimated that more than 3 million people currently have COPD, and an estimated 2 million people have COPD which remains undiagnosed because they do not visit physician due to absence of apparent respiratory problems in them and/or most patients mainly with mild-to-moderate disease, are thought to remain undiagnosed or not diagnosed until they are in their fifties [22,23].

Apart from the destruction of alveoli and smaller airways, cigarette smoking induces changes in the larger airways, with impairment of ciliary function and hypertrophy of mucosal glands in the bronchi [2]. These findings are consistent with the earlier report, which shows that smoking reduces endogenous production of nitric oxide [6]. This is due to the reduction in the activity of the enzyme nitric oxide synthase, which is caused by an inadequate supply of the coenzyme tetrabiopterine [24]. Hence, the enzyme is present, but is unable to produce nitric oxide. In view of the above-mentioned facts, it is reasonable to assume that increasing the production of endogenous nitric oxide should improve the pulmonary functions in smokers. Therefore, it was aimed to increase the production of endogenous nitric oxide by stimulating the activity of NOS enzyme.

In the present study, some common vegetables, fruits and spices listed in [Table/Fig-2] are shown that produce nitric oxide in vitro. Now, it raises a question, whether these diet also increase the production of nitric oxide endogenously. Indeed, in the present study, garlic supplementation in smokers has significantly increased the endogenous production of nitric oxide [Table/Fig-3] and also significantly improved the impaired pulmonary function tests of smokers [Table/Fig-4,5]. These findings are supported by the earlier report that nitric oxide synthase activation is a unique mechanism of garlic action [25,26].

The most likely explanation is that supplementation of garlic, which increases endogenous production of nitric oxide, might be doing so by stimulating nitric oxide synthase, a membrane bound enzyme. Though, reports lack studies regarding the effects of garlic on smokers’ pulmonary function test parameters, it is well reported with the therapeutic health benefits of garlic explaining the possible reason for enhanced pulmonary functions mentioning that increased endogenous production of nitric oxide causing pulmonary vasodilatation and also has a modulatory role of the airway defense system affecting the beating frequency of cilia [4,27]. According to a study carried out at the Jiangsu Provincial Center for Disease Control and Prevention in China that people who ate raw garlic at least twice a week had a 44% lower risk of developing lung cancer [28]. Throughout history in the Middle East, East Asia and Nepal, the beneficial effects of the garlic supplementation have been reported in several studies to treat several lung diseases including common cold, bronchitis, tuberculosis, inflammatory disorders of the lungs and several other diseases with explaining the possible mechanism by decreasing the leukocyte inflammatory mediators [29-31].

Thus, garlic may offer relief from airway restriction and impaired lung function by improving the clearance of mucus from the airways, help airways relax, and improve airflow into the lungs. These could be the possible explanation for uplifting the reduced pulmonary function test parameters in chronic smokers after garlic supplementation in our study.

Interestingly, exclusively garlic supplementation in our study has improved pulmonary functions significantly in chronic smokers, which seems promising in its medical effects on respiratory system, yet to be confirmed by further detailed studies. This study would have been strongly conclusive if larger numbers of samples had been included. Moreover, it would be more clarifying if we could study the animal model with histological changes in respiratory system brought about by garlic supplementation.

In conclusion, we can say that dietary supplementation of garlic significantly improved pulmonary functions in smokers. Therefore, it can be suggested that garlic should be incorporated in common food items daily and may reduce the risk factors for causing impaired pulmonary function in smoker. However, keeping in mind the deleterious effects of smoking of tobacco in the form of cigarettes, an anti smoking awareness should be launched in the society.