3.1. Comparison of microwave oven and convective oven drying for determination of moisture content in herbs
Accurate and rapid MC determination of fresh or dried plant tissues and their powders is essential for research laboratories and herbal industries. MC determination is important to control shelf-life as well as compositional analysis of herbal products. Often the prescribed method of MC determination by various regulatory agencies is by calculating the weight loss of a sample on (complete) drying at 105ºC in a hot air oven [22, 23]. However, the method has two major limitations, firstly longer time of drying for complete removal of moisture and secondly broader intra-sample variation. In this study, four medicinally valuable plant samples were dried by two different methods; by microwave drying at 720W power and by well-known hot air oven at 105ºC. Both the processes were compared for complete moisture removal. Indeed, the drying rate in the domestic microwave oven was one order of magnitude faster. Figure 1 indicates that the moisture diffusivity in all the samples under microwave drying was about ten times more than that in the hot air oven drying at 105ºC.
In the hot air oven, complete removal of moisture from all four samples took at least 7 h whereas in microwave oven it took less than 16 minutes. Considering the hot air oven method as a gold standard, the moisture content determined for four medicinal plants viz, CA, EGF, ENF and MN using microwave oven was compared with the moisture content determined by hot air oven. The Pearson and Spearman correlation coefficient between the MC determined by both the methods were respectively 0.9 and 0.76 (Table 1).
Table 1
Moisture content comparison of different samples in 105ºC oven drying and Microwave drying (720W)
105ºC Oven drying | Microwave (720W) |
0.89 | 0.89 |
0.89 | 0.89 |
0.87 | 0.87 |
0.87 | 0.87 |
0.89 | 0.84 |
0.89 | 0.84 |
0.72 | 0.77 |
0.70 | 0.77 |
This finding supports that the microwave oven drying is suitable for MC determination of herbal leaves like hot air oven. The method is further examined for the intra-sample variation. MC of an herbal sample (CA) was measured ten times with both the methods and the dispersion of the MC data was compared with a Box-whisker plot (Fig. 2).
The origin of variation in a hot air oven was studied and the heterogeneity of air flow rate and temperature in different locations of the hot air oven was recorded. The samples kept at various trays faced different local air conditions and therefore suffered different extents of drying. It is well known that hot air ovens without forced convection will have spatial heterogeneity of temperature. However, to identify the extent of spatial temperature and air flow distribution, laboratory hot air ovens with forced convection were studied and surprisingly all of them showed significant non-uniformity. The extreme air velocities in two different locations inside the dryer cabinet was observed at 0.1 and 3.6 m/s. Whereas the lowest and highest temperature seen inside the same cabinet was 95ºC and 107ºC. Differences of 10ºC and flowrate differences of one order of magnitude can considerably under-dry or over-dry the samples. Tables 2 and 3 indicates the heterogeneity of temperature and airflow rate inside the dryer cabinet used for the present study. It can be anticipated that the designs of laboratory hot air ovens may not always ensure uniform airflow. Many times, finer mistakes in the design of such ovens may create poor mixing and therefore poor temperature control. Computational fluid dynamics can be used to optimize the design of a hot air oven for narrow temperature and air velocity distribution within the cabinet [24]. Another issue we could identify is solid loss due to high airflow rate in hot air ovens especially for friable materials like leaves, flowers etc. Such issues are less likely in presently available domestic microwave ovens. Though standing wave pattern of microwave inside the oven and dielectric heterogeneity of plant tissues cause some non-uniformity in heating of the sample volume [25]. Ceramic/Stainless steel enamel cavity, appropriate cavity geometry, controlled frequency and turntable plate for holding the samples make most of the domestic microwave oven suitable for uniform heating of plant materials. Therefore, for small volume of samples (5–25 g) uniformity of microwave ovens are superior than that of hot air oven. In this study also microwave oven showed significantly lower intra-sample variation compared to hot air oven (Fig. 2). Moreover, in terms of quality and yield, it is often anticipated that at high temperature essential oil of many medicinal plants may deteriorate. Therefore, distillation method of MC determination is often preferred for such medicinal plants with high oil content [22]. The method is problematic due to difficulty in recovery of the condensate and cleaning the distillation set up.
Table 2
Spatial distribution of temperature and air flow rate in hot air oven
OD (105°C) | TD (50°C) |
Cabinet locations | Flow rate(m/s) | Temperature (°C) | Cabinet locations | Flow rate(m/s) | Temperature (°C) |
Near Fan | 2.9 ± 0.09 | 107 ± 1.73 | Middle Right (Near Fan) | 3.6 ± 0.15 | 51.2 ± 0.66 |
Up Middle | 0.7 ± 0.06 | 103 ± 1.73 | Middle Middle | 0.8 ± 0.09 | 50.6 ± 1.18 |
Down Middle | 1.5 ± 0.09 | 97 ± 1.45 | Middle Left | 0.5 ± 0.09 | 48.9 ± 1.24 |
Up Right | 0.7 ± 0.15 | 94 ± 2.03 | Top Right | 0.4 ± 0.06 | 48.2 ± 0.66 |
Down Right | 1.2 ± 0.15 | 95 ± 1.15 | Top Middle | 0.3 ± 0.06 | 47 ± 1.36 |
| | | Top Left | 0.1 ± 0.03 | 46.7 ± 0.49 |
Down Right | 1.7 ± 0.20 | 42.6 ± 1.24 |
Down Middle | 1.3 ± 0.15 | 43.2 ± 1.07 |
Down Left | 0.3 ± 0.06 | 42.8 ± 1.01 |
Infront of Door | 0.08 ± 0.01 | 42 ± 1.13 |
OD: Oven dried; TD: Convective tray dried; Results were expressed as mean ± standard error of triplicate runs. |
Also, the formation of emulsions may be hard to be separated and assessed for oil and moisture content. For fresh plant tissues still, the loss on complete drying is suitable method considering the higher moisture content (70–95%) compared to the oil contents. The oil concentration in plant parts are typically less than 10% except some seeds. However, the commercial Karl Fischer tilt rotors costs couple of thousand USD and often a complex system for regular use in academic or industrial labs. Even 1% moisture content in a standard 5 g sample will cause 0.05 g of change which is achievable for all practical purposes. In case of microwave drying, general concern is volatilization of solids in a sample leading to overestimation of moisture content [26].
Table 3
Spatial distribution of temperature in microwave oven (720W)
MW (720W) | 1 min | 2 min | 3 min | 4 min | 5 min | 6 min | 7 min | 8 min | 9 min | 10 min |
Left Boundary | 72 ± 0.88 | 74 ± 0.33 | 71 ± 0.58 | 71 ± 0.33 | 73 ± 0.67 | 72 ± 0.33 | 72 ± 0.33 | 72 ± 0.58 | 74 ± 0.58 | 73 ± 0.88 |
On Plate | 99 ± 0.52 | 100 ± 0.39 | 100 ± 0.33 | 103 ± 0.58 | 104 ± 0.33 | 105 ± 0.58 | 106 ± 0.58 | 105 ± 0.88 | 104 ± 0.33 | 105 ± 0.33 |
Right Boundary | 93 ± 0.58 | 97 ± 0.58 | 96 ± 0.58 | 95 ± 0.58 | 90 ± 0.58 | 93 ± 0.58 | 96 ± 0.58 | 97 ± 0.88 | 92 ± 0.88 | 95 ± 0.88 |
Upper | 112 ± 0.61 | 104 ± 0.62 | 105 ± 0.41 | 107 ± 0.41 | 105 ± 0.62 | 107 ± 0.62 | 105 ± 00.62 | 103 ± 0.41 | 103 ± 0.62 | 109 ± 0.62 |
MW: Microwave dried; Results were expressed as mean ± standard error of triplicate runs. |
Following reaction shows the carbonization (charring) of sugars. For each gram of hexose (equivalent) about 0.6 g of weight loss happens.
$${C}_{12}{H}_{22}{O}_{11}\to 11{H}_{2}O +12C$$
We simulated the effect of different percentages (0.5–50%) of volatilization and also the effect of different degrees (0.5–50%) of incomplete drying on the estimated moisture content of a sample (Table SI1 and Table SI2). The standard deviation of all the simulated moisture content values was 0.322%. Therefore, the correct rounding off-figure for representing the moisture content determination was considered upto one decimal place [27]. For the same accuracy of MC values, it was found that with less than 5% volatilization or 5% lesser drying, the MC values does not change significantly for a sample irrespective of its MC. Hence, an herbal product with a sugar/hexose content as high as 8% will show insignificant error in MC even with complete carbonization. Generally, fresh herbal crops like flowers, leaves, rhizomes etc. contain 70–95% moisture. Same simulation showed that no error can be detected in MC determination even if there is 15% under drying or 15% solid volatilization for any sample with more than 70% MC. Therefore, when MC to be determined for fresh (expected MC > 70% in wet basis) and dried (expected MC < 10% in wet basis) herbal substances microwave oven-based method might fail if the thermo-labile compounds respectively
Table 4
Order of time to reach the equilibrium moisture content of herbal samples
Sample Name | Drying time to reach EMC | Initial moisture content (%, wb) | Equilibrium moisture content |
TD (50°C), h | MW (720W, 90%), minutes | FD (-40ºC), h | TD (50°C), h | MW (720W, 90%), minutes | FD (-40ºC), h |
CA | 8 | 13 | 48 | 89.8 | 1.8 | 1 | 0 |
ENF | 12 | 16 | 48 | 89.6 | 0.2 | 2 | 0 |
EGF | 8 | 9 | 48 | 87.5 | 0.9 | 0 | 1 |
MN | 9 | 15 | 48 | 77.9 | 1 | 0 | 1 |
CA: Centella asiatica; EGF: Eryngium foetidum; ENF: Enhydra fluctuans; MN: Marsilea minuta; TD: Convective tray dried; MW: Microwave dried; FD: Freeze dried; EMC: Equilibrium moisture content; wb: wet basis |
crosses 15% and 5% considering 5 g of sample size. The thermo-labile compounds may be essential oils, sugars or other heat-sensitive compounds. In addition to non-uniform energy distribution, another reason for the lack of repeatability was found to be heterogeneity of samples. Many a times fresh herbal samples may contain different tissues (e.g., stem and leaves together) which will have different drying behaviour and moisture content.
3.2. Drying characteristics of four herbs in three different types of dryer and properties of their powder
In a separate set of experiment, microwave drying was compared with two drying methods; a commercially popular drying method i.e., convective tray drying (at 50ºC) and the other one is a peer accepted sophisticated drying technique, i.e. freeze drying. Convective drying is very common form of drying in industries. However, it is a slow and poor drying process in terms of bioactives. On the other hand, freeze drying is a costly process to be adopted for common usage. In the present trial, microwave drying was acceded to check its suitability to prepare dried herbs for further used as herbal beverages (decoction and infusion), ingredients for curries or cuisines or component of herbal medicine preparation (e.g., hydroalcoholic extract) and quality analysis of herbal products. For the three drying methods, moisture content of the sample was estimated for different time intervals till the constant weight of the sample was reached. The order of time to reach the equilibrium moisture content was microwave drying (8–12 min) < low temperature convective drying (50ºC) (9–16 h) < freeze drying (36–48 h) (Table 4). As expected, the drying time was dependent on the herb variety since the surface area and thickness of the leaf and the residual stem as well as the internal cell structures, were different. EGF and ENF took respectively fastest and slowest time to reach EMC in both convective and microwave drying. For freeze drying, we could take weight change only after every 12 h and all the four samples reached EMC between 36–48 h (Fig. SI1). Since the sublimation and subsequent diffusion rate of water molecules in sub zero temperature was much slower than that duirng drying at 50ºC or higher temperature, freeze drying took longest time to dry. However, in all three methods the equilibrium moisture content was quite low and the highest EMC was observed for Enhydra fluctuans i.e., 2% in case of microwave drying (Table 4). This indicates that microwave drying is the fastest method for drying herbs to achieve a desired moisture content. For consistent results (sensory quality, bioactivity etc.) of an infused beverage, soup, sprinkle, medicinal decoction etc. herbs are often used in a powdered form where its soluble components get extracted in the (aqueous) solvent. Both qualitative and quantitative extraction of the molecules depends on particle size as well as its porosity of the dried herbal powders. Specific drying technique i.e., moisture removal mechanism, temperature of drying and rate of moisture removal of the herbal product controls not only the composition of the molecues (degradation or derivatization of the small compounds) but also their milled powder characteristics (particle size and porosity). From the sieve analysis of the powders obtained from four different plant
Table 5
Particle size distribution of herbal powders
Sample Name | Average particle size (µm) (2000-63) | Average particle size (µm) (250 − 63) |
TD (50°C) | MW (720W) | FD (-40°C) | TD (50°C) | MW (720W) | FD (-40°C) |
CA | 716.48 ± 6.48 | 663.76 ± 2.52 | 735.02 ± 0.39 | 305.33 ± 0.89 | 276.89 ± 0.53 | 287.17 ± 0.79 |
ENF | 707.65 ± 13.58 | 676.25 ± 0.97 | 688.34 ± 0.17 | 309.60 ± 4.28 | 302.09 ± 0.59 | 295.19 ± 0.87 |
EGF | 641.77 ± 16.59 | 660.17 ± 2.74 | 694.13 ± 1.76 | 295.22 ± 5.99 | 294.51 ± 1.13 | 301.43 ± 1.13 |
MN | 614.25 ± 5.80 | 633.16 ± 5.95 | 661.41 ± 2.01 | 292.11 ± 1.66 | 292.09 ± 3.21 | 304.33 ± 0.04 |
CA: Centella asiatica; EGF: Eryngium foetidum; ENF: Enhydra fluctuans; MN: Marsilea minuta; TD: Convective tray dried; MW: Microwave dried; FD: Freeze dried; Results were expressed as mean ± standard error of triplicate runs. |
leaves dried with three different types of drying methods but milled under same conditions showed that the particle size distribution was not greatly influenced by the method of drying but varied with the type of the plant material (Table 5).
Table 6
Comparative study of different methods of drying
Sample Name | TD (50°C) | MW (720W) | FD (-40°C) |
Bulk Density (kg/m3) | Tapped Density (kg/m3) | Apparent Porosity (%) | Bulk Density (kg/m3) | Tapped Density (kg/m3) | Apparent Porosity (%) | Bulk Density (kg/m3) | Tapped Density (kg/m3) | Apparent Porosity (%) |
CA | 125.00 ± 00 | 163.33 ± 3.33 | 18.00 ± 2.00 | 236.89 ± 7.01 | 283.70 ± 2.01 | 34.04 ± 0.24 | 217.49 ± 4.73 | 246.95 ± 3.05 | 29.63 ± 0.37 |
ENF | 270.47 ± 7.31 | 313.73 ± 19.61 | 25.10 ± 1.57 | 272.73 ± 12.99 | 312.50 ± 0.00 | 34.38 ± 3.12 | 226.0 ± 1.28 | 266.71 ± 3.56 | 23.97 ± 2.35 |
EGF | 200.08 ± 4.00 | 227.74 ± 10.35 | 24.95 ± 1.14 | 215.08 ± 2.31 | 253.21 ± 3.21 | 32.88 ± 2.12 | 221.0 ± 1.22 | 241.0 ± 2.90 | 26.54 ± 2.73 |
MN | 233.99 ± 4.11 | 285.95 ± 8.17 | 22.88 ± 0.65 | 289.92 ± 4.20 | 305.36 ± 2.33 | 30.54 ± 0.23 | 241.28 ± 8.72 | 283.82 ± 6.04 | 22.71 ± 0.48 |
CA: Centella asiatica; EGF: Eryngium foetidum; ENF: Enhydra fluctuans; MN: Marsilea minuta; TD: Convective tray dried; MW: Microwave dried; FD: Freeze dried; Results were expressed as mean ± standard error of triplicate runs. |
Usually with decreasing moisture content, there should be a decrease in bulk density as well as the tap density of the dried powder but we could observe the density values varied according to the method of drying and not just the moisture content [28, 29]. Among all the dried materials, the highest bulk density powder was of Marsilea minuta (289.9 kg/m3) when dried in the microwave oven (Table 6). However, the lowest bulk density powder could be seen for Centella asiatica (125.0 kg/m3) when dried with convective drying at 50ºC. Also the apparent porosity i.e., the ratio of tapped density to true density of the dried powder from microwave was higher than that of freeze drying and lowest in low temperature convective drying (50ºC). Generally, tapped density is more for regular shaped particles like sphere cubes etc., than irregular shaped particles like needle shaped or fibrous one. In the present study, the tapped density does not follow a specific trend and the lowest and highest tap densities were found to be for Centella asiatica and Enhydra fluctuans. Owing to the difference in their tissue structure they gave rise to particles of different shapes as well as pore pattern. Also the structural components like lignin, cellulose, hemicellulose and pectin can vary greatly for CA, EGF, ENF, and MN which control the true density of the leaf powder particle specifically in dried form. Since lignin and cellulose have less affinity to moisture than hemicellulose and pectin which influence the drying behaviour greatly. Such difference in composition reflects in the particle size. For all four plants depending on the method of drying, 30–50% of the powdered samples are in the range of 250 µm to 106 µm. Etti et al.[30] and Fitzpatrick et al.[31] reported that all the pure herbal powders with particle sizes below 100 µm made them cohesive. Hence, the tiny particles will form interparticular binding to make their flow difficult [32]. Since particle size distribution and porosity dictates its functional properties; adsorption-desorption behaviour, extractability of solubles from the particles, enzymatic digestion, shelf-life, compaction and flowability etc. microwave drying can be used as a better method than low temperature convective drying or even freeze drying to obtain dried powder with better porosity and smaller particle size.
3.3. Effect of three different drying on extractability of alcohol soluble components and antioxidant activity of the resultant extract
In terms of better retention of bioactive compounds in a herb, freeze-drying is generally considered as the most reliable method for drying for their processing and analysis. In this study, we wanted to confirm two possibilities; whether the drying method can influence the matrix structure and therefore total extractability of a specific soluble (e.g., methanol) fraction in terms of TSS and any specific functional property like antioxidant activity of the millieu of molecules in the extract. The TSS of the four-leaf samples listed in the Fig. 3a were dried in three different drying methods. For extraction, methanol was used since most primary and secondary metabolites are soluble in methanol and it has good penetration into the cells. All the four leaves dried using microwave and freeze dryer had a 10–20% higher amount of TSS compared to convective tray dried sample at 50ºC. However, there is no significant difference found between freeze-dried and microwave dried samples of EGF, ENF and MN. In CA, we have found 18% higher TSS than freeze-dried samples. Nevertheless, the comparable amount of TSS of the microwave with freeze-dried showed that the drying method did not alter the porous matrix of the herbs. Singh et al.[33] also reported that microwave (0.03–0.78) and freeze (0.03–0.88) dried samples have more or less the same porosity for potato, whereas in the case of convective drying, it is in the narrow range of 0.03 to 0.20. Apart from TSS, the so-called total phenolic content in terms of gallic acid equivalent of methanolic extract was similar in both microwave and freeze drying, on the other hand, low temperature convective dried samples had 25–45% lesser TPC values (Fig. 3b). The total flavonoid content of the methanolic extracts from different dried samples of the four plants followed almost same pattern as TPC (Fig. 3c). However, the antioxidant capacity in terms of Fe3+ reduction (IRP), free radical scavenging (ARSA) and lipid peroxidation inhibition (LPOX) out of the four herbs, CA and ENF are in the same order and showed high antioxidant potential (Fig. 3d-3f) whereas other two plants had significantly lesser antioxidant potential. Therefore, particular antioxidant capacity (e.g., Fe3+ reduction or free radical scavenging capacity) of the plants were dependent on both the herb variety and the methods of drying. Potisate et al.[34] reported that approximately 43–62% increase in quercetin and kaempferol levels in the M. oleifera leaves after drying in a freeze dryer(-85ºC) as well as in microwave (150, 450, 900W) compared to dried in a convective dryer at 50ºC, also a reduction in phytochemicals was observed at 60ºC in convective tray dryer since multiple reaction can take place at that high temperature. Similarly, Max industrial microwave did a comparative study on M. oleifera leaves and reported an increase in both micronutrients and macronutrients on microwaving. There is an increase in sulphur (0.72%), iron (16.5%), copper (14.7%), manganese (10.2%), vitamin C (46.7%), vitamin E (33.02%), vitamin B2 (47.9%) and folate (14.3%) compared to convective tray dryer at 40ºC whereas, there is a minimum decrease in phosphorus (5%), potassium (7%), calcium (6.7%), zinc (1.6%) and vitamin A (6.6%) content. Such findings support that not only both microwave and freeze drying is superior than low temperature convective drying but also quality of microwave dried leaves is closer to freeze dried w.r.t. extractability and antioxidant properties of the herbs.
3.4. Effect of drying on polyphenol oxidase
To strengthen the conclusion of section 3.3 that microwave and freeze-dried herbs had similar TPC and TFC and both values were higher than convective dried condition, we assayed the native polyphenol oxidase assay for all four leaves. Figure 4 shows that out of four types of drying, microwave dried samples lost the PPO enzymatic activity similar to that of 105ºC whereas in all four leaves the PPO could survive in significant amount in both freeze-drying and low temperature convective drying at 50ºC. This supports the fact that at a low temperature drying there is enough PPO enzymatic activity that for the long period of drying the oxidative enzyme might degrade some of the phenolic compounds present in the leaves. However, it is important to know the magnitude of oxidase enzymes activity and how it would affect the degradation of phenolics within the leaves.
Because during freeze drying the PPOs might not react in sub-zero condition (-40ºC) even if high residual PPO activity is traced in the dried product. Depending on the variety, the activity level varied between the drying methods. For example, EGF showed more PPO activity in freeze dried samples but ENF showed more activity in 50ºC dried sample. According to Loh et al.[35] as polyphenol oxidase activity increased, the percentage loss in TPC also increased; however, there was no obvious trend observed for the peroxidase enzyme. Reports also stated that enzymatic activities generally decreases with decreasing water activity [36]. It was observed that all the dried leaves (aw between 0.1–0.3) have low water activity (Table SI3). Lin et al.[37] reported that the release of active oxidative enzymes (PPO and POD) could cause enzymatic degradation and therefore reduce the number of extractable phenolics when leaf tissues were damaged. As PPO enzymes are involved in the production of quinones from monophenols and diphenols, it is logical to expect that high PPO activity would degrade more phenolics in the plant leaf samples [35]. Moreover most plant tissues contain isoenzymes of PPOs with different substrate affinity and thermal stablity. This is probably the reason in different drying condition specific polyophenols may be present or absent. Microwave dried leaves of Catharanthus roseus, Backhousia citriodora, G. pseudochina, M. communis contained higher concentration of gallic acid, caffeic acid, rutin, myricetin 3-o-rhamnoside, quercetin 3-o-glucoside, quercetin 3-o-rhamnoside, myricetin, quercetin, kaempferol, and chlorogenic acid [38–41]. Therefore, further investigation of polyphenol distribution under different drying conditions will provide valuable insights into the impact of processing methods on the phytochemical composition of the herbs.
3.5. Effect of drying on polyphenols of selected four herbs using HPLC
The signature chromatogram profiles of the extracts from CA, EGF, ENF and MN leaves dried with freeze-drying and microwave and convective drying processes are shown in Table SI4 (a) – SI4 (d). The normalized HPLC chromatogram confirmed that the freeze-dried and the microwave dried samples contained higher amounts of phenolics than those in the convective dried samples and for most compounds (identified as well as unidentified peaks) the general order of concentration in all four herbs was freeze drying > microwave drying > low temperature convective drying. Moreover, there was an increased concentration of chlorogenic acid, rutin, ethyl protocatechuate, naringin, and myricetin
recorded in the microwave, as well as freeze-dried CA leaves over convective drying. Whereas apigenin was not identified in microwave dried sample of CA but its concentration was much higher in freeze-dried sample than the convective dried one. Others have also reported superiority of microwave and freeze drying for polyphenol extraction/retention in several plant materials [42–44]. Apart from altering leaf powder, particle microstructure and PPO enzyme inactivation, microwave drying can cause releasing of free phenolic compounds from the bound phenolic compounds [45–47]. For example, caffeic acid one of the intermediate in lignification can exist as caffeoyl shikimic acid, caffeic aldehyde, and caffeoyl alcohol which may not be the extractable. These compounds can be transformed during microwave heating and become extractable [43, 47, 48]. In this study, both freeze-drying and microwave drying could retain more phenolic compounds from the CA, ENF, EGF, and MN. Also, similarity analysis with Pearson correlation coefficient and Euclidian distance of the four plants showed that microwave dried samples are close to freeze dried samples (Table 7).
Table 7
Similarity indices of the three types of drying on HPLC spectra of four types of herbal plants
Name Sample | Similarity marker | TD (50°C)_MW (720W) | MW (720W)_FD(-40°C) | TD (50°C)_FD(-40°C) |
Pearson correlation coefficient | Euclidean distance | Pearson correlation coefficient | Euclidean distance | Pearson correlation coefficient | Euclidean distance |
CA | Area (mAU*s) | 0.05 | 1224.0 | 0.97 | 241.2 | 0.07 | 1181.9 |
Height (mAU) | 0.11 | 162.2 | 0.98 | 29.1 | 0.09 | 149.8 |
ENF | Area (mAU*s) | 0.44 | 2772.5 | 0.87 | 1919.5 | 0.14 | 4091.9 |
Height (mAU) | 0.51 | 181.1 | 0.79 | 156.1 | 0.14 | 278.2 |
EGF | Area (mAU*s) | -0.17 | 1929.0 | 0.84 | 906.7 | -0.09 | 1433.9 |
Height (mAU) | -0.16 | 113.0 | 0.74 | 63.0 | -0.12 | 100.9 |
MN | Area (mAU*s) | 0.15 | 577.8 | 0.92 | 304.8 | 0.03 | 781.8 |
Height (mAU) | 0.01 | 111.7 | 0.92 | 52.2 | -0.12 | 146.6 |
CA: Centella asiatica; EGF: Eryngium foetidum; ENF: Enhydra fluctuans; MN: Marsilea minuta; TD: Convective tray dried; MW: Microwave dried; FD: Freeze dried |
Nevertheless, according to the area under the curve and height of the HPLC chromatogram for both identified and unidentified compounds, it was observed that most of the compounds present in the samples are increasing due to microwave drying. Therefore, microwave drying can be a fast-drying method for isolating a good amount of bioactive compounds from these selected plant species.
3.6. Chemical profiling of one of the selected herbs for similarity of drying process
LC-MS analysis was performed for three different drying methods to identify phenolic compounds present in Centella asiatica methanolic extracts. A total of 31 compounds were identified in any of the three dried extracts, which belonged to 9 different classes: caffeoylquinic acids, hydrocinnamic acid, tannins, flavonoids, terpenoids, stilbene, and fatty acids (refer to Table 8). The results showed that 19% of these compounds were present in all three types of drying. When compared to freeze-dried, the convective-dried sample shared 27% common compounds, while microwave-dried samples showed 46% common compounds in Centella asiatica extract. Furthermore, microwave-dried samples had 38% compounds in common to convective-dried samples. All the identified compounds had been previously reported in different Centella asiatica extracts. For instance, the study identified five phenolic acids known as caffeoylquinic acids (CQAs), which had been previously reported in Centella asiatica by Long et al.[49]. In addition to phenolics, the flavonoid naringin was detected in all three drying methods, whereas luteolin-7-glucuronide and quercetin were only found in convective drying and microwave drying. A significant group of compounds, including madecassoside, oleuropein, asiaticoside, and madecassic acid, were detected in all three drying processes. However, compounds such as 3,5-di-o-caffeoylquinic acid, piceatannol, p-coumaric acid, ferulic acid derivative, chlorogenic acid, and 3,4-dicaffeoylquinic acid were exclusively found in microwave-dried and freeze-dried Centella asiatica extracts. Moreover, tannins like trigalloylquinic acid were detected in all three drying methods. It is important to note that this untargeted metabolomics approach only represents a small fraction of the metabolome, and a collection of chromatographic methods is
Table 8
Chemical profiling of Centella asiatica methanolic extracts
Sl. No. | Rt (min) | Parent ion (m/z) | MS/MS fragments | Tentative identity of compound | TD (50ºC) | MW (720W) | FD (-40ºC) | Compound Class |
1. | 2.46 | 343 | 153, 201 | Piceatannol | × | ✓ | ✓ | Stilbene |
2. | 2.68 | 632 | 128, 341, 539, 632 | Oleuropein | ✓ | ✓ | ✓ | Terpenoids |
3. | 21.76 | 1033 | 546, 740, 973 | Madecassoside | ✓ | ✓ | ✓ |
4. | 22.79 | 1017 | 179, 538.3, 740, 957 | Asiaticoside | ✓ | ✓ | ✓ |
5. | 24.80 | 713.2 | 134, 375.2, 585.1 | Asiatic acid | ✓ | ✓ | × |
6. | 27.79 | - | 503 | Madecassic acid | ✓ | ✓ | ✓ |
7. | 3.79 | 180.1 | 130.2 | Caffeic acid | × | ✓ | × | Hydrocinnamic acid |
8. | 7.32 | - | 164.2 | P-coumaric acid | × | ✓ | ✓ |
9. | 28.56 | - | 143, 293 | Cinnamoyl-glucose | ✓ | × | × |
10. | 22.05 | 1018 | 193.1, 538.6, 887 | Ferulic acid derivative | × | ✓ | ✓ |
11. | 12.67 | - | 113, 203.2 | Chlorogenic acid | × | ✓ | ✓ | Caffeoylquinic Acids |
12. | 13.36 | 707 | 191, 353, 467, 707 | 1,5-Di-O-Caffeoylquinic acid | × | × | ✓ |
13. | 16.95 | 515 | 335, 191.1, 161 | 3, 4-Dicaffeoylquinic acid | × | ✓ | ✓ |
14. | 23.94 | 1060 | 113, 375.4, 565.5, 740.8, 1000 | Neochlorogenic acid | × | ✓ | × |
15. | 18.29 | 1031.7 | 161.1, 353, 515.4, 615.4 | 3, 5-Di-o-caffeoylquinic acid | × | ✓ | ✓ |
16. | 18.60 | - | 285, 461.1 | Luteolin-7-glucoronide | ✓ | ✓ | × | Flavonoids |
17. | 19.60 | 565.5 | 489.3, 511.4, 565.3 | Naringin | ✓ | ✓ | ✓ |
18. | 21.36 | 737.6 | 713, 723 | Quercetin derivative | ✓ | ✓ | × |
19. | 16.07 | 601 | 161, 233 | Eriodictyol 7-(6-galloylglucoside) | × | × | ✓ |
20. | 20.20 | 587 | 187, 429, 489 | Catechin derivative 1 | ✓ | × | × |
21. | 20.77 | 713 | 333.1, 429, 489 | Catechin derivative 2 | ✓ | × | × |
22. | 25.73 | - | 285.1 | Kaempferol | ✓ | × | × |
23. | 36.97 | - | 277.2 | Myricetin | ✓ | × | ✓ |
24. | 33.04 | - | 281 | Linoleic acid | × | × | ✓ | Fatty acid |
25. | 33.74 | 837.6 | 279.2 | Linolelaidic acid | ✓ | × | × |
26. | 34.13 | 695 | 205, 249.2, 389, 452, 695 | Palmitic acid | ✓ | × | ✓ |
27. | 33.07 | 737 | 105, 213, 273, 391, 487 | Pelargonidin isomer | ✓ | ✓ | × | Anthocyanidin |
29. | 18.08 | 613 | 161, 353, 477, 515, 613 | Trigalloylquinic acid | ✓ | ✓ | ✓ | Tannins |
30. | 25.30 | - | 127 | Pyrogallol | × | × | ✓ | Other polyphenols |
31. | 35.09 | 815 | - | Unknown | × | × | ✓ | |
Rt: Retention time; TD: Convective tray dried (50ºC), MW: Microwave dried (720W); FD: Freeze dried (-40ºC) |
necessary for a comprehensive understanding of the global metabolome.
The results also indicated the preferential distribution of certain compounds in convective-dried, microwave-dried and freeze-dried Centella asiatica methanolic extracts.