Multiple pathways are responsible for Anti-inflammatory and Cardiovascular activities of Hordeum vulgare L.

World literature, especially, Greek, Roman and Egyptian literatures point to the dietary and medicinal properties of Hordeum vulgare L. (HV) [1]. HV, a member of the grass family, Poaceae, is an annual crop used throughout ancient civilizations as an important food source. The genus Hordeum consists of 32 species and 45 taxa. HV belong to a single biological species, which is an annual and is diploid. All varieties of HV have hollow stems in the forms of cane, produced from fibrous roots. There is a spike-shaped arrangement of seed at the end of each stem.

Until the 19^th century, barley was the major part of diet of millions of people. Even now, it is eaten as such or in processed form, in various quantities at various ontogenic stages by people of all income and age groups globally as functional food. Various clinical and pre-clinical studies have confirmed the positive and beneficial effects of barley on human health. Consumption of barley which contains many medicinally active phytocompounds is usually associated with improvement in health. Use of barley can protect against diabetes, obesity, atherosclerosis, cancer, stroke and insulin resistance [2].

A number of pharmacological activities of HV have been reported in the literature. Hot water extracts of HV protect from damage due to oxidation of various proteins [3]. Anti-apoptotic activity of the HV has been reported and is attributed to the presence of 3,4 dehydroxybenzaldehyde, which protects from DNA damage [4]. Lipopolysaccharide-induced inflammation is also inhibited by methanolic extract of the aerial parts of HV, both in vitro and in vivo[5]. Barley or HV is also used to treat a number of diseases in traditional Ayurvedic system of medicine in India, especially in the treatment of urinary stones [6]. The growth of the urinary stones [7] as well as mild hyperuricemia [8] was found to be inhibited by using the seed extracts of HV.

The main constituents of HV include important antioxidants such as vitamin E, phytic acid, selenium, tocotrienols, and various phenolic acids. After the consumption of HV, these antioxidants are released at differential rate throughout the gastrointestinal tract over a long period of time [9]. Health benefits of HV consumption are numerous [10] and are mainly attributed to various mineral, dietary fibres, unsaturated fatty acids and sphingolipids, in addition to the antioxidants and vitamins [11]. Apart from that a number of other antioxidant compounds have been isolated from HV [12].

HV is used by traditional healers to treat various inflammatory and cardiovascular diseases, without the knowledge of pharmacologic rationale behind its actions [6]. Despite the very important roles of platelets activation in the development of acute thrombosis and cardiovascular diseases (CVD), no data are available concerning the effect of barley on platelet activation thrombus formation. Since a number of plants have been reported to display inhibition of arachidonic acid (AA) metabolism and platelet aggregation [13]-[18], we therefore, screened various fraction of HV against cyclooxygenase (COX) and lipoxygenase (LOX) pathways of AA metabolism. Since both of these enzymatic pathways are implicated in oxidative stress, we further evaluated the actions of HV on superoxide dismutase (SOD) and glutathione peroxidase (GPx). These activities have not been previously reported for HV.

The chemicals used in the study were purchased from BDH (acetic acid and citric acid), Merck (sodium phosphate-mono and dibasic), Amersham Biosciences (¹⁴C arachidonic acid) and Sigma (the remaining), all from USA and were of analytical grade.

Crude extract from HV was unable to inhibit ADP-induced platelet aggregation; however, collagen and PAF-induced aggregations were blocked partially while the most potent antiplatelet effect was observed against AA-induced-platelet aggregation. The dose of the extract showing 50% inhibitory effect against platelet aggregation (mean IC₅₀ value in μg) against various agonists were 18.33 ± 3.51 for AA, 187.71 ± 19.57 for PAF, 211.23 ± 27.92 for collagen and 15.88 ± 2.14 for aspirin (positive standard control) (Table 1 and Figure 1). Crude extract of HV was also more effective against COX-mediated pathway of AA metabolism compared to LOX-mediated pathway. IC₅₀ (mean ± SD in μg) values were 24.34 ± 5.33 for TXB₂, 78.45 ± 12.29 for LP1 and 89.44 ± 16.22 for HETE. Crude extract potently elevated SOD activity (EC₅₀ in μg, 32.88 ± 3.19) but was ineffective in increasing GPX activity up to 1 mg dose (Table 2).

Aqueous fraction of HV was unable to inhibit PAF and ADP-induced platelet aggregation, however, AA and collagen -induced aggregations were blocked partially. The dose of the fraction showing 50% inhibitory effect against platelet aggregation (IC₅₀ value in μg) against various agonists were 58.73 ± 12.82 for AA, 121.55 ± 20.32 for PAF (Table 1 and Figure 1). Aqueous fraction of HV was ineffective against LOX-mediated pathway of AA metabolism while partially blocking COX-mediated pathway. IC₅₀ (mean ± SD in μg) values were 74.67 ± 12.52 for TXB₂, while no significant effect was observed on LP-1 and 12-HETE up to a dose of 1 mg. Aqueous fraction just like the crude extract potently elevated SOD activity (EC₅₀ in μg, 28.21 ± 4.73) but was ineffective in increasing GPX activity up to 1 mg dose (Table 2).

Chloroform fraction of HV, just like the crude extract, was unable to inhibit ADP-induced platelet aggregation; however, collagen and PAF-induced aggregations were blocked partially while the most potent antiplatelet effect was observed against AA-induced-platelet aggregation. The dose of the fraction showing 50% inhibitory effect against platelet aggregation (IC₅₀ value in μg) against various agonists were 21.63 ± 4.10 for AA, 98.56 ± 16.68 for PAF, 126.73 ± 22.36 for collagen (Table 1 and Figure 1). Chloroform fraction of HV was also more effective against COX-mediated pathway of AA metabolism compared to LOX-mediated pathway. IC₅₀ (mean ± SD in μg) values were 28.63 ± 6.76 for TXB₂, 66.52 ± 10.51 for LP1 and 118.44 ± 19.75 for HETE. Chloroform fraction just like the crude extract and aqueous fraction potently elevated SOD activity (EC₅₀ in μg, 28.83 ± 5.48) but was ineffective in increasing GPX activity up to 1 mg dose (Table 2).

n-hexane fraction of HV was unable to inhibit collagen and ADP-induced platelet aggregation, however, AA and PAF-induced aggregations were blocked partially. The dose of the fraction showing 50% inhibitory effect against platelet aggregation (IC₅₀ value in μg) against various agonists were 86.74 ± 12.54 for AA, 97.82 ± 15.63 for PAF (Table 1 and Figure 1). LOX pathway of AA metabolism was blocked potently while COX metabolite was partially inhibited. IC₅₀ (mean ± SD in μg) values were 40.34 ± 7.46 for TXB₂, 37.61 ± 4.71 for LP1 and 60.52 ± 4.60) for HETE (Figure 2). n-hexane potently elevated the activities of both SOD and GPx with EC₅₀ (μg) of 35.29 ± 5.33 and 23.17 ± 3.29 respectively (Table 2).

Despite cardiovascular protective potential of available synthetic drugs, these drugs have various side-effects like skin rashes, bleeding, headaches and gastrointestinal problems. Therefore research is now directed towards green pharmaceuticals (medicines of plant origin) which are believed to be free from side-effects. Plants have been used for centuries to obtain treatment for various human diseases. One of the most important therapeutic targets for treating cardiovascular and inflammatory disease is the inhibition of AA metabolites through COX [28]-[30]. There are reports suggesting natural inhibition of AA metabolites [31]. Dual inhibition of LOX and COX pathways by natural compounds is also known and thought to be contributed by the secondary metabolism of plants [32],[33]. Natural inhibitors of both inflammation and oxidative stress, however, are less common but could be of great therapeutic importance. Here in this report, we show that HV strengthens the antioxidant defense system and inhibits LOX and COX pathways of AA metabolism.

In the present investigation, crude extract of HV showed potent inhibition of AA-induced platelet aggregation, was partially effective against PAF and collagen while no significant effect was observed against ADP. These results suggest that crude fraction inhibits platelet aggregation mainly acting through COX pathway as AA induces platelet aggregation after conversion to TXA2 by platelet COX enzyme. Inhibition of thromboxane-mediated platelet aggregation, however, seems to be a common mechanism shared by many other plants [3],[15]-[17],[34]. Further experiments with AA metabolism also suggest that crude fraction blocks COX mediated pathway of AA as it significantly inhibited TXB2 production from AA while both metabolites of LOX pathway were only partially inhibited. Crude extract was devoid of any significant effect on GPx, however, SOD activities were strongly elevated by the application of crude extract.

Both COX and LOX enzymatic pathways are implicated in oxidative stress and studies indicate that inhibition of COX [26] as well as LOX [35],[36] reduces oxidative stress. Our results show that crude extract of HV as well as all three fractions elevated SOD activity while only n-hexane fraction enhances GPx activity. Previous studies also suggest that inhibition of COX lowers the oxidative stress [37]. Rise in SOD activity are likely to be a consequence of COX inhibition which can up regulates antioxidant enzymes such as SOD [38]. Similarly COX-2 and GPx are inversely related [39] and the effect of HV in this case may be related to its ability to inhibit COX. A number of studies show the presence of antioxidant potential of medicinal plants [32],[33] including increasing activities of SOD and GPx [26].

Aqueous fraction of HV did not show any significant activity against platelet aggregation with only partially inhibiting AA and collagen while having no significant effect on PAF and ADP. Interestingly aqueous fraction had no effect on LOX mediated metabolism of AA while only partial inhibition of TXB₂ was observed suggesting aqueous fraction has less of compounds responsible for AA metabolism. This result also explains relatively low effect of aqueous fraction on AA-induced platelet aggregation. Just like crude extract, aqueous fraction showed strong elevation of SOD activity but ineffective against GPx. Consistent with previous studies [40],[41], these results suggest that aqueous fraction has concentrated compounds that have potent effect on SOD and that the fractionation process has resulted in the distribution of compounds responsible for COX inhibition into other fractions as well.

Chloroform fraction of HV showed activities similar to the crude extract and akin to previous reports [42],[43], strongly inhibited TXB₂ production and AA-induced platelet aggregation and having significant effect on elevating SOD activities. This suggests that chloroform fraction retains the activities contained in the crude extract and that fractionation process did not affect much in the distribution of phytocompounds in chloroform fraction. This also means that SOD elevation activity of HV is probably exhibited by more than one compound because aqueous fraction also had similar effects on SOD. Chloroform fraction of other plants has been shown previously to possess activities against platelet aggregation [44].

The pattern of activities observed with n-hexane fraction of HV was slightly different and exhibited strong effect against LOX-mediated metabolites of AA and significantly elevated GPx activity. These two activities are not shared by any other fraction, nor by the crude extract. Similar activities were previously found in n-hexane fraction of other plants [45],[46]. This suggests that crude extract might contain ingredients that do not allow compounds responsible for these activities to show any significant effect and act as their blockers.

Metabolism of AA, especially by two enzymatic pathways-LOX and COX produces various eicosanoids which play crucial role in inflammation [47]. Therefore, all the diseases with underlying inflammation are likely to benefit from inhibition of AA metabolism [48]. Since inflammation is associated with weakening antioxidant defense, the results of our study show that HV that possesses phytocompounds with anti-inflammatory as well as anti-oxidant potential can be used in such circumstances. Our study signifies the importance of HV as a starting point for the development of novel compounds against two of the most important enzymes implicated in inflammation in addition to enhancing activities of SOD and GPx. It is possible that separate compounds are responsible for activities related to COX, LOX, SOD and GPx; however, further, studies may also reveal that two or more of the above activities are possessed by a single phytocompound. Further characterization of HV and its fractions and isolation of pure compounds could provide us valuable information in this context.

Our study demonstrates that HV possesses activities against all human platelet agonists used in this study except ADP, and inhibited both COX and LOX pathways of AA metabolism. It also elevated the activities of SOD and GPx. However, these activities were distributed in various fractions. Therefore, it is unlikely that a single phytocompound is responsible for all these activities. Some fractions of HV showed more potent activities than others while certain fractions were almost completely devoid of anti-inflammatory effect (aqueous). Interestingly, crude extract was unable to enhance GPx activity while n-hexane fraction which was obtained from the fractionation of crude extract, caused significant elevation of GPx activities.

SG and SA conceptualized and performed the study, NK and QTZ analyzed the data, NBT and AH did literature search, HZEJ, MM and MZ did data analysis and manuscript writing. All authors read and approved the final manuscript.