2.1 Saliva parameters
2.1.1 Saliva flow rate (SFR)
As shown in Fig. 1(a), there was no significant changes in salivary flow rates of the resting status (S1), 3 min (S2) and 30 min (S3) after drinking water or black tea (p>0.05), although the SFR increased mildly after drinking both liquids. It indicates neither tea nor water significantly stimulate the salivary secretion. The maximum SFR was found 30 minutes after ingestion in all groups. The highest average SFR (0.433 mL/min) was observed in assessors drinking hot water, while the highest individual SFR (1.499 mL/min) was found in an assessor drinking the cold tea. Within each testing group/stage, a large individual difference was found, which is consistent with previous reports 15,27. Some participants constantly showed either much higher or much lower SFR levels than the average, no matter what sample they drank. For example, volunteer No.10 and No.17 always reported the lowest SFRs, while volunteer No.13 and No.20 always showed the highest.
In general, the unstimulated whole salivary flow rate ranges from 0.25 to 0.90 mL/min with a mean value of approximately 0.4 mL/min, which could be stimulated by vision, smell or taste or parasympathetic nerve activity occurs 28,29. The mean value of resting SFR was 0.384 mL/min in this study, which is consistent with the previous studies. However, a greater number of maximum SFR was reported here. It is known that the SFR is correlated to the size of the individual's salivary glands, which is particularly pronounced in stimulated SFR 27. In addition, the SFR of female volunteers might be elevated during their the menstrual cycle 30. The reason why we are observing a relatively big variation between individuals in this study remains unclarified. The greater volume or higher concentrations of black tea infusion, the different method or timing of saliva collection, and the longer period of drink intervention may be needed to further verify the above observations.
Usually, chewing or drinking food can cause an increase in salivary flow rate. Consumption of solid foods and chewing show the more dominant effects on salivary secretion than the beverages and drinking 27. The gum chewing increases SFR by 2-5 times 31. Our results demonstrate that neither water nor the black tea infusion immediately promote the saliva secretion in healthy adults.
In addition, the temperature of oral exposure may affect saliva secretion. The transient membrane potential cation receptor melatonin 8 (TRPM8) is a cold receptor in the oral cavity that can be stimulated by cold and menthol 32, and the stimulation of cold receptors leads to increased salivary flow rate 33. As reported by Lee et al., the parotid gland saliva flow was stimulated at 10℃, but not at 22℃ and 44℃ 34. Brunstrom et al. found that cold water (at 3℃) significantly increased salivary flow rate compared to the warmer water (at 13℃, 23℃, 33℃) 26. The cold water (at 8℃) didn’t induce a higher SFR than the hot water (at 57℃), which could be attributed to the wider time gap between drinking and saliva collection than the Brunstrom’s report (after 3 min or 30 min v.s. immediately after ingestion), or to the different method of saliva collection (spontaneous saliva flow from the whole oral cavity v.s. a cotton swab sit underneath the tongue).
2.1.2 Total protein content of saliva (TPC)
The influence of ingesting different samples on the total protein content of saliva is presented in Fig. 1(b). TPC ranged from 0.39 to 2.26 mg/mL. Compared with the corresponding water groups, the TPC of assessors drank the hot tea and cold tea increased right after the ingestion, although insignificantly (p>0.05), and further increased significantly after 30 minutes of ingestion (p<0.05). Moreover, the salivary protein level slightly decreased 3 minutes after cold water ingestion (p>0.05) and significantly increased 30 minutes after cold water ingestion (p<0.05). The ingestion of hot water did not change the TPC.
The secretion of protein and fluid from salivary gland is regulated by parasympathetic nerve and sympathetic nerve impulses. The parasympathetic nerve impulses produce high-flow and low-protein saliva, while the sympathetic nerve impulses produce low-flow and high-protein saliva 35. As shown in Fig. 1(a) and 1(b), the rather similar SFR in all groups and the significantly elevated TPC in the tea ingestion group indicates the black tea ingestion may possess more potent impacts on the sympathetic nerves. The high salivary TPC accompanied by low SFR was observed in assessor No.8/10/12. Assessor No.20, however, exhibited low salivary TPC and high SFR. In addition, the proteins expressed by different salivary glands vary greatly 36. For example, the serous fluid from parotid gland does not contain mucin but is rich in amylase and proline-rich proteins (PRP). Black tea contains rich number of tannins, which readily bind to salivary proline-rich proteins. As a result, the anti-nutritional effects of tannins, such as growth retardation and impaired nutrition absorption 37, could be mitigated. The protein compositions of saliva after tea ingestion need to be further studied to reveal their salivary glands origin.
The study showed a relatively stronger promotion in TPC by ingesting the hot tea than the cold tea, while the hot water exhibited the weaker influences on TPC than the cold water. In Eccles' study, the cold stimulation (temperature) to the oral receptors leads to elevated saliva secretion 38 and a higher level of salivary proteins. It is known that the whole-body and continuous exposure to cold environment (i.e., at 4℃ for 15 min) significantly increases the salivary flow rates and protein concentration 39, whilst in this study, a short (in seconds) and partial (mouth only) exposure to cold drink (at 8℃) only induced a mild increase in the salivary proteins and no significant changes in the saliva secretion.
2.1.3 Total antioxidant capacity (FRAP)
As shown in Fig. 3(c), the black tea ingestion tended to increase the FRAP although not significantly (p>0.05). The salivary FRAP was reduced immediate after drinking hot water (p>0.05) and cold water (p>0.05) and resumed to the initiate level (by hot water, p>0.05) or an increased level (by cold water, p<0.05). The delayed effect of cold-water ingestion (30-min after) on FRAP echoed well with the effect of cold black tea ingestion, implying the cold drink may possess more profound impacts on salivary FRAP than the hot drink.
The effects of food fluids on human saliva FRAP was less reported. Villaño et al. found that ingestion of 500 mL of oolong beverage contained oolong tea extract (2.4 g/L) significantly increased plasma FRAP in healthy subjects within 1 hour to 4 hours 40. The study of Wiseman et al. and Langley also found that drinking green tea and black tea increased the plasma FRAP of subjects, respectively 41,42. In the study of Chong et al., drinking Tieguanyin and black tea did not significantly change the salivary FRAP, while intake of vine tea enhanced the antioxidant capacity of saliva over a longer period of time, which were related to the high level of polyphenols and flavonoids in vine tea 24. Azimi et al found that drinking green tea effectively boosted the antioxidant capacity of smokers' saliva in a short run and a long term 43.
2.1.4 Salivary uric acid concentration (UA)
As shown in Fig. 1(d), the black tea ingestion, either hot or cold, significantly reduced the salivary uric acid level 3 min after the ingestion (p<0.05), which rose back to the initiate level after 30 minutes. The temperature of tea soup had little influence. Both hot water (58℃, 200 mL) and cold water (7℃, 200 mL) significantly increased salivary uric acid levels (p < 0.05) 30 min after the water ingestion. The results were in line with the previous studies reported by Chong et al. 24. The salivary UA level in this study remained in the normal range of healthy adults.
Uric acid is believed to be the dominant antioxidant compound in saliva, as it accounts for about 70% of the total antioxidant capacity 44. Recent studies confirmed a positive correlation between circulating UA and salivary UA 45,46. However, the extraordinary high level of salivary uric acid is associated with body fat accumulation, liver steatosis and increased incidence of psychological disorders 47–49. The external factors can rapidly alter salivary uric acid concentration. Lucas et al. reported that acute social stress rose the adults’ salivary UA level rapidly and significantly 50. In another study, the cold pain stimulus at 5℃ rose salivary UA level in healthy young adults about half an hour after the stimulation, demonstrating a delayed response to acute physical stress 51. As in this study, the ingestion of cold or hot water caused a delayed increase in the salivary UA levels, too.
Little was known on the effect of black tea consumption on salivary uric acid. The effects of black tea extract on plasma uric acid in mice has been highlighted in multiple studies 52,53. The black tea extracts reduced plasma uric acid in hyperuricaemic mice to a higher extent than the green tea extracts, which is mainly due to the EGCG inhibition of xanthine oxidase (XO) and adenosine deaminase (ADA) in liver, thereby reducing uric acid synthesis 54. On the other hand, tea catechins and theaflavin can regulate uric acid transport proteins to inhibit plasma uric acid excretion 55. It remains unclear whether the black tea induced acute decrease in salivary UA could be attributed to the same mechanism.
2.1.5 Salivary thiol content (SH)
The thiol content of saliva reveals the status of oxidative stress 15. As shown in Fig. 1(e), regardless of hot or cold, the SH increased significantly right after the tea ingestion (p<0.05) and still remained higher than the resting level after 30 min (p>0.05). Ingestion of water did not cause any significant changes in SH level (p>0.05). As the concentration of salivary thiol did not coordinate with the gradual increasing trend of total protein, it is proposed that the proteins are not the major source of SH.
Glutathione (GSH) plays a crucial role in mitigating oxidative stress 56. Smoking can cause a decrease in salivary thiol levels 16,17. The decreased level of salivary GSH was found in the patients of oral squamous cell carcinoma, oral leukoplakia 57, Alzheimer's disease, and many other chronic degenerative diseases 58.
Black tea has been found to effectively reduce oxidative damage in mice by improving plasma GSH and SOD levels 59,60. The immediate increase of salivary thiol levels reported here may contribute to the elevated FRAP in saliva and provides a fast-responding biomarker for assessing the antioxidant activity of food components.
2.1.6 Salivary malondialdehyde content (MDA)
Oxidative stress causes lipid peroxidation and induces the formation of various products, including malondialdehyde 61,62. As shown in Fig. 1(f), drinking hot black tea immediately elevated the salivary MDA level by 16.39% (S2, p<0.05) to 2.201 µM, while the cold tea showed the same trend but not significantly. Both hot water and cold water ingestion suppressed the salivary MDA significantly by 24.74% and 26.23%, respectively (p<0.05), demonstrating neither temperature nor water fraction have dominant effects on salivary MDA.
In general, extraordinary high levels of salivary MDA are often associated with oral inflammation and metabolic diseases 63–65. In a study, the salivary MDA content of healthy individuals aged 20-39 was 1.87 ± 0.68 μM 66, which is consistent with our results. Freese et al. found that green tea extract significantly reduced plasma malondialdehyde (MDA) concentration of healthy females 67. The long-term effects of black tea on salivary MDA had rarely been studied. Sun et al. found that the black tea polysaccharide significantly mitigated the CCl4-induced increase in liver MDA in mice 59. As reported previously, the same kind of black tea increased the salivary MDA 15, while the oolong tea and vine tea took longer time (30 min) to raise the salivary MDA level 24. The MDA increments caused by the tea ingestion is temporary and well below the pathological increments. It becomes a curious but maybe meaningful subject how the black tea will affect the salivary MDA of patients of oral inflammation and metabolic diseases. Furthermore, the relation between the immediate and long-term changes of salivary MDA in response to black tea ingestion would help us to better understand the human physiological influences of tea, thus needs to be clarified by further studies.
2.2 Correlation analysis
Pearson correlation and principal component analysis were used to reveal the correlation of salivary parameters in 20 subjects. The production rate is referring to the amount of substance produced as a function of time. The concentration of salivary compositions or the FRAP were converted to production rate and tabulated in Table 2. Table 3 explains the factor loadings for the analyzed variables from F1 to F5. A biplot of the principal components (F1 and F2) is shown in the Fig. 3. The large loadings of TPC, FRAP, SH and MDA contribute to F1, while F2 is related to UA. The SFR has a similar loading in F1 and F2.
The correlations among salivary parameters at different stages were studied by Pearson’s correlation and correlation coefficients were presented in Fig. 2. The values marked in bold indicate a high statistical correlation between the two variables. At stage 1, a strong positive correlation was clearly shown between TPC and SH. At stage 2, the ingestion of both black tea and water led to multiple changes in the correlations: SH was highly and positively correlated with FRAP and MDA, but no longer showed strong positive correlation with TPC; there was a significant positive correlation between MDA and TPC; the strong negative correlations were found between UA and FRAP, SH, and MDA. In stage 3, there is no significant correlation between parameters except for the significantly negative correlation between TPC and SFR, and the correlation in stage 2 decreases or changes, reflecting the difference between the immediate and delayed effects of water and black tea consumption on saliva variables. Furthermore, salivary TPC levels in stage 3 continued to increase due to black tea consumption, and other saliva parameters were close to the values of stage1, which affected the correlation of stage 3.
In addition, as shown in Figure 2(d), water ingestion weakened the positive correlation between SH and TPC, while strengthened the positive correlation between UA and FRAP. However, tea consumption led to a significant increase in saliva SH and MDA and a significant decrease in UA, which contributed to the positive correlation between the two and their negative correlation with TPC and UA, respectively. Black tea consumption increases the oxidative stress of saliva, and a large increase in SH may contribute most of the antioxidant capacity of saliva, compensating for the decrease in UA.
As shown in Fig. 3, principal component 1 and 2 account for 81.93% of the total variation in the dataset. The distribution of observed values (blue dots) is shown in a PCA biplot. The variables (red lines) are distributed in the first and second quadrants, where TPC, SFR and UA are in the first quadrant and FRAP, MDA and SH are in the second quadrant. The objects of water at S2 (salivary attributes immediate after water ingestion) distributed in the same quadrant and stay on the same side (left) of diagram with objects of stage1, and are not related to the variables. It elucidates water does not affect saliva immediately. When observing the immediate effects of black tea consumption, there is a strong positive correlation between the salivary thiol and tea drinking, either hot or cold. The black tea ingestion almost immediately elevates salivary MDA and FRAP regardless the temperature. Thirty minutes after the ingestion, salivary SFR and TPC are more positively correlated with the delayed effect of black tea or water. The uric acid may be less important in this study, squared cosines of salivary uric acid belongs to F2.