US 200400.5881.8A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2004/0058818A1 Li (43) Pub. Date: Mar. 25, 2004

(54) SYSTEM FOR INCREASING THE (60) Provisional application No. 60/161,460, filed on Oct. PRODUCTION OF CAMPTOTHECIN FROM 25, 1999. PLANTS Publication Classification (76) Inventor: Shiyou Li, Nacogdoches, TX (US) (51) Int. Cl." ...... A01N 25/00; AO1B 79/02 Correspondence Address: (52) U.S. Cl...... 504/116.1; 47/58.1 R Alan R. Thiele (57) ABSTRACT JENKENS & GILCHRIST The present invention provides a System for increasing the 3200 Fountain Place production of indole and quinoline alkaloids, particularly 1445 ROSS Avenue camptothecins and related compounds, in plants, based upon Dallas, TX 75202-2799 (US) management of plant hormones. This invention includes a System and a method for accelerating the growth of young (21) Appl. No.: 10/670,930 tissues, inducing the production of camptothecins-bearing glandular trichomes, maximizing the harvest of glandular (22) Filed: Sep. 25, 2003 trichomes, and preserving and treating the glandular tri chomes for maximum camptothecins yield. The present Related U.S. Application Data invention is useful for any plant matters containing alka loids, regardless of the plant matters genetic origin, natural (63) Continuation of application No. 09/696,042, filed on or cultivated variety, and natural or cultivated growing Oct. 25, 2000. conditions. Patent Application Publication Mar. 25, 2004 Sheet 1 of 37 US 2004/0058818A1

1 R=R=H, R=OH 10 2 R=R=OH, R=H 3 R=OCH R=H.R=OH 4 R=H, R=R=OH 5 R=H, R=OCH R=OH 6 R=R=R=H 7 R=R=H, R=O(CH2)CH 8 R=OCH R=H, R=O(CH2)CH

Diagrams of the chemical structures of major natural camptothecin and its analogs in Camptotheca acuminata: camptothecin (1), 10-hydroxycamptothecin (2), 10 methoxycamptothecin (3), 11-hydroxycamptothecin (4), 11 methoxycamptothecin (5), 20-deoxycamptothecin (6), 20 hexanoylcamptothecin (7), 20-hexanoyl-10 methoxycamptothecin (8), 22-hydroxyacuminatine (9), 19 hydroxymappicine (10), 19-O-methylangustoline (11), and vincoside-lactam (12). Fig. 1. Patent Application Publication Mar. 25, 2004 Sheet 2 of 37 US 2004/0058818A1

Intermediate Tissue Leaf Definition 1-4 week old

CPT% (fw) 0.02.2450- 0.00135 Stem Definition = 2 year old

CPT % (fw) 0.00795 - 0.00036

Stem Wood Definition = 2 year old CPT % (fw) 0.00309 - 0.00007 0.00566 0.00027

Note: Shiyou Li, et al. unpublished.

CPT distribution in different tissues of Camptotheca acuminata (Seed source: SFA 94-03; leaf, stem, and root materials were collected in May, wood and bark samples were collected in August and fruit samples were collected in June, August, and October, respectively) (mean + S.d.) (on the basis of fresh weight). Fig. 2 Patent Application Publication Mar. 25, 2004 Sheet 3 of 37 US 2004/0058818A1

a. Top Scanning electron micrograph of surface view of lower leaf epidermis of Camptotheca Lowreyana Katie' Scale Bar = 100 um. b. Bottom) Scanning electron micrograph of mature glandular trichome (GT) on lower leaf surface of Camptotheca Lowreyana Katie'. Scale Bar - 5um. Fig. 3 Patent Application Publication Mar. 25, 2004 Sheet 4 of 37 US 2004/0058818A1

Average Average Average Glandular Young Leaves Old Leaves Species/Variety Glandular trichome Density CPT Concentration CPT Concentration

trichome length trichome width (um) (% s.d.) (% + s.d.) un cauma 34.87 - 3.92 13.64 - 1.97 46.80 E 5.26 0.05822 - 0.01654 0.016 07 at 0.00204

yunnanensis 35.20 - 2.70 13.72 it 1.27 27.10 - 1.40 0.05443 - 0.01499 0.1308 - 0.002.7

Yalal 46.49 4.5 10.94 - 1.42 81.50 - 6.50 0.084.23 - 0.01541 0.020210,0321 lowreyana 42.46 - 3.34 14.64 - 1.68 65.50 E 10.41 0.10641 - 0.01612 0.0274 - 0.00167 "Katie'

56.32 it 6.72 4.02 - 1.87 69.20 - 14.45 0.12284 + 0.01089 0.02632 + 0.00278 "Hicksii' Note: Shiyou Li, et al. unpublished.

Glandular trichome size and density on lower leaf surfaces and CPT concentration in leaves of Camptotheca (on the basis of fresh weight.)

Fig. 4 Patent Application Publication Mar. 25, 2004 Sheet 5 of 37 US 2004/0058818A1

COOH COOH

N NH 2 ------ee-ee-o-o-o-o-o-wo- Cu?N tryptophanH (Trp) Route A H Indoleacetic acid (IAA)

tryptamine

N NH 21 O-glucose

N - O CHOC StrictOsidine Camptothecin (CPT)

Diagram of two biosynthetic pathways showing tryptophan (TRP) as a biosynthetic precursor for both indoleacetic acid (Route A for stimulating growth) and camptothecin (Route B for inhibiting growth).

Fig. 5 Patent Application Publication Mar. 25, 2004 Sheet 6 of 37 US 2004/0058818A1

W.

Photograph of C 2 tothec 2 l O W e 22. "Katie Fig. 6 Patent Application Publication Mar. 25, 2004 Sheet 7 of 37 US 2004/0058818A1

Drawing of a Camptotheca leaf after leaf-tip pinching.

Fig. 7 Patent Application Publication Mar. 25, 2004 Sheet 8 of 37 US 2004/0058818A1

control

before pruning second pruning third pruning fourth pruning first harvest

1.

Drawing of a Camptotheca Seedling T-pruning treatments and control. Fig. 8 Patent Application Publication Mar. 25, 2004 Sheet 9 of 37 US 2004/0058818A1

Drawing of the leaf-tip pinching technique as applied in Camptotheca. Fig. 9 Patent Application Publication Mar. 25, 2004 Sheet 10 of 37 US 2004/0058818 A1

Patent Application Publication Mar. 25, 2004 Sheet 11 of 37 US 2004/0058818A1

quºu?e3.J.L.Quoqo?I Patent Application Publication Mar. 25, 2004 Sheet 12 of 37 US 2004/0058818A1

Treatment I Treatment II Control (cm) (30 cm) (40 cm) Before treatment (March 25, 1997) 52.61 - 544 52.15 3.57 50.72 - 6.12 After treatment

(March 25, 1997) 52.61 - 5.44 30.00 - 0.00 40.00 - 0.00 Net Growth

(March 25-July 12, 1997) 40.10+ 8.86 a 39.599.73 a 32.93 - 7.82 b Net Growth

(July 12-Sept. 13, 1997) 19.52 - 11.39 a 31.87+ 8.41 b 32.00 - 9.52 b

Mean height growth of plants with different T-pruning treatments (mean + s.d.) (means with the same letter are not significantly different at O=0.05).

Fig. 12 Patent Application Publication Mar. 25, 2004 Sheet 13 of 37 US 2004/0058818A1

Treatment I Treatment II

Control (30 cm) (40 cm) Before treatment (March 25, 1997) 1.03 + 0.17 1.04 - 0.27 1.03 - 0.17 After treatment

(March 25, 1997) 1.03 + 0.17 1.00 0.00 OO at 0.00 July 12, 1997 10.143.06 a 10.21 it 3.60 10.04 - 3.16 Sept. 13, 1997 - - 10.56 - 3.34 a 17.00 - 5.82 b 16.33 - 5.21 b

Mean branch number of plants with different T-pruning treatments (mean -- s.d.) (means with the same letter are not significantly different at o=0.05).

Fig. 13 Patent Application Publication Mar. 25, 2004 Sheet 14 of 37 US 2004/0058818A1

Camptotheca acuminata Monthly Biomass Production of intact Young Tissues -Control - T-Pruning

ea 92 t

d S

s E 9.

March April May June July Aug Sept Oct Nov Month

Graph of the monthly biomass production of intact young tissues with and without T-pruning

Fig. 14 Patent Application Publication Mar. 25, 2004 Sheet 15 of 37 US 2004/0058818A1

Treatment I Treatment II Control (30 cm) (40 cm)

CPT Content (%) 1.0164 - 0.00141 a 0.0351 - 0.0020 b .0437 - 0.0037 c

Effects of T-pruning treatments on CPT contents (%) of intact young tissues of Camptotheca acuminata (mean E s.d.) (means with the same letter are not significantly different at or=0.05) (samples were collected on June 20, 1998) (fresh weight).

Fig. 15 Patent Application Publication Mar. 25, 2004 Sheet 16 of 37 US 2004/0058818A1

Camptotheca acuminata Effect of Pinching on CPT Concentration Whole Plant

SS

O

ho d ()

O U) H al d

3 4. 5 Days after Pinching

Graph of the effect of pinching on CPT concentration in the whole plant of Camptotheca acuminata.

Fig. 16a Patent Application Publication Mar. 25, 2004 Sheet 17 of 37 US 2004/0058818A1

Camptotheca acuminata Effect of Pinching on CPT Concentration intact Young Tissues

a S. 9. wd t ed

C O 9 H U

3 4. 5 Days after Pinching

Graph of the effect of pinching on CPT concentration in intact young tissues of Camptotheca acuminata.

Fig. 16b Patent Application Publication Mar. 25, 2004 Sheet 18 of 37 US 2004/0058818 A1

Camptotheca acuminata Effect of Pinching on CPT Yield Whole Plant

G E. O o d He f 9

3 4 5 Days after Pinching

Graph of the effect of pinching on CPT yield on the whole plant in Camptotheca acuminata.

Fig.16c Patent Application Publication Mar. 25, 2004 Sheet 19 of 37 US 2004/0058818A1

Camptotheca acuminata Effect of Pinching on CPT Yield Intact Young Tissues

S 9. 9. () und U wd S s O) O ha O 5 9. g X H al C)

3 4. 5 Days after Pinching

Graph of the effect of pinching on CPT yield on intact young tissues in Camptotheca acuminata.

Fig.16d Patent Application Publication Mar. 25, 2004 Sheet 20 of 37 US 2004/0058818A1

Camptotheca acuminata CPT induction by Pinching TYoung Leaves Relatively Young Leaves

SS o t s d

C C D o O

Days after Treatment

Graph of CPT induction by pinching in young leaves and relatively young leaves of Camptotheca acuminata.

Fig. 17a Patent Application Publication Mar. 25, 2004 Sheet 21 of 37 US 2004/0058818A1

Camptotheca acuminata CPT Induction by Pinching Old Leaves

s SS w

s C O D Ho (9

3 4. 5 Days after Treatment

Graph of CPT induction by pinching old leaves of Camptotheca acuminata.

Fig. 17b Patent Application Publication Mar. 25, 2004 Sheet 22 of 37 US 2004/0058818A1

Camptotheca acuminata CPT induction by Pinching Yound Stems - A - Old Stems

s Š o 9 wd h wd

s O U H U

3 4 5 Days after Treatment

Graph of CPT induction by pinching in young stems and old stems in Camptotheca acuminata.

Fig. 17c Patent Application Publication Mar. 25, 2004 Sheet 23 of 37 US 2004/0058818A1

Camptotheca acuminata CPT induction by Pinching A Yound Roots O Old Roots

S. C O d - S yed

U O () H f D

2 3 4 5 Days after Treatment

Graph of CPT induction by pinching in young roots and old roots in Camptotheca acuminata.

Fig. 17d Patent Application Publication Mar. 25, 2004 Sheet 24 of 37 US 2004/0058818A1

Pruning only 1.025061- 0.00389

Pruning + Pinching 0.03043 - 0.00129

Effects of pinching treatments on CPT contents (%) of intact young tissues of Camptotheca acuminata under irrigation system (mean + s.d.) (means with the same letter are not significantly different at C=0.05) (samples were collected on August 30, 2000) (fresh weight).

Fig. 18 Patent Application Publication Mar. 25, 2004 Sheet 25 of 37 US 2004/0058818A1

Light Levels Sample Size Height Living Branch

... " Number Full Sunlight 52.78 - 13.08 a 1.12 - 0.33 a

Mean growth of one-year-old seedlings grown under different light levels (mean + s.d.) (means with the same letter are not significantly different at O=0.05) (data were collected on July 12, 1998.

Fig. 19 Patent Application Publication Mar. 25, 2004 Sheet 26 of 37 US 2004/0058818A1

Light Levels Sample Size Height Living Branch Glandular Trichome Number Density (no./mm2) Full Sunlight 377.89 - 59.99 17.05 - 5.08 52.16 110.61 - 21.92 b 3.67 - 146 78.23 b

Mean growth and glandular tricome density of three-year old seedlings grown under different light levels (mean + s.d.) (means with the same letter are not significantly different at C-0.05) (data were collected on July 15, 1998.

Fig. 20 Patent Application Publication Mar. 25, 2004 Sheet 27 of 37 US 2004/0058818A1

Treatment Biomass (g) (fw) | CPT Concentration (%) (fw) CPT yield (mg)

Natural Dry 74.60 1774 0.05041 - 0.00940 a 37.2112 - 9.8481 a Condition

Under Irrigation 213.29 + 39.15 b 0.02754 000648 b 59.2765. 22.007 1 a

Production of biomass and CPT of intact young tissues under different water conditions (mean ts.d.) (means with the same letter are not significantly different at C(=0.05) (data were collected on August 30, 2000.

Fig. 21 Patent Application Publication Mar. 25, 2004 Sheet 28 of 37 US 2004/0058818A1

Relatively Young Young Stems Intact Clipping Leaves Biomass (g) (fw) 2.2485 - 0.1039 a 7.735 - 0.506 b 2.8950 - 1.6334 12.8950 - 3789 CPT Content (%) fw) 0.0380 at 0.0053 a 0.0214 - 0.003 b 0.0080 00030 c 0.0203 - 0.0008 CPT Yield (mg 0.8504 - 0.0806 a 0.1002 + 0.1007 c 2.6054 + 0.1804

Distribution pattern of biomass, CPT content, and CPT yield in an intact clipping (mean + s.d.) (means with the same letter are not significantly different at CF0.05) (Samples were collected on May 4, 2000).

Fig. 22 Patent Application Publication Mar. 25, 2004 Sheet 29 of 37 US 2004/0058818A1 Biomass Yield CPT Yield

(g) (fw) (mg)

Total CPT Yield: 2.6 mg/Intact Clipping Fig. 23 Patent Application Publication Mar. 25, 2004 Sheet 30 of 37 US 2004/0058818A1

Camptotheca acuminata Effect of Harvest Cycle on CPT Content (%) of intact Young Tissues

3 4. 5 Harvest Cycle (weeks)

Graph of the effect of harvest cycle on CPT content of intact young tissues in Camptotheca acuminata.

Fig. 24a Patent Application Publication Mar. 25, 2004 Sheet 31 of 37 US 2004/0058818 A1

Camptotheca acuminata Change of CPT Yield with Harvest Cycle

S N S w C C. his OL S S. s X

s

3 4. 5 Harvest Cycle (weeks)

Graph of the effect of harvest cycle on CPT yield of intact young tissues in Camptotheca acuminata.

Fig. 24b Patent Application Publication Mar. 25, 2004 Sheet 32 of 37 US 2004/0058818A1

Camptotheca acuminata CPT Variation of Young Leaves with Tree Age

0.12

0.1

0.08

0.06

0.04

O.02

1 2 3 4 5 6 7 8 Tree Age (years)

Graph of the variation in CPT concentration of young leaves with tree age in Camptotheca acuminata.

Fig. 25 Patent Application Publication Mar. 25, 2004 Sheet 33 of 37 US 2004/0058818A1

Camptotheca acuminata Monthly Change of CPT Content (%) of Intact Young Tissues

s wn C O) wind C O O s CO

March April May June July Aug Sept Oct Nov Month

Graph of the monthly change of CPT content of intact young tissues of Camptotheca acuminata.

Fig. 26a Patent Application Publication Mar. 25, 2004 Sheet 34 of 37 US 2004/0058818A1

Camptotheca acuminata Monthly CPT Yield

16 5 d g > He ? O

March April May June July Aug Sept Oct Nov Month

Graph of the monthly yield of CPT of intact young tissues of Camptotheca acuminata.

Fig. 26b Patent Application Publication Mar. 25, 2004 Sheet 35 of 37 US 2004/0058818A1

Preservation Method CPT Content (%+s.d.)

Oven-dry (65°C) 0.02715 - 0.0061

CPT Preservation of intact young tissues preserved by different methods (samples were collected on May 31, 2000) (6 replications, fresh weight).

Fig. 27 Patent Application Publication Mar. 25, 2004 Sheet 36 of 37 US 2004/0058818A1

Camptotheca acuminata Effect of Homogenizer Treatment on CPT Extraction Determination of Treatment Time

s ha Cl O "O C w t X L

20 40 60 80 100 120 140 Treatment Time (seconds/10 g fresh sample)

Graph of the effect of homogenizer treatment on CPT extraction in Camptotheca acuminata by duration of treatment time.

Fig.28a Patent Application Publication Mar. 25, 2004 Sheet 37 of 37 US 2004/0058818A1

Camptotheca acuminata Effect of Homogenizer Treatment on CPT Extraction Extraction Efficacy

s S. H L. U) O C wd s Extraction under Reflux s ma vid x A Homogenizer Treatment

One TWO Three Four Five Extraction Times

Graph of the effect of homogenizer treatment on CPT extraction in Camptotheca acuminata by extraction efficacy.

Fig. 28b US 2004/0058818A1 Mar. 25, 2004

SYSTEM FOR INCREASING THE PRODUCTION 0012 D. Ophiorrhiza (Rubiaceae); OF CAMPTOTHECIN FROM PLANTS 0013 E. Merrilliodendron (Icacinaceae); REFERENCE TO RELATED APPLICATION 0014) F. Mostuea (Loganiaceae); 0001. This application claims priority from U.S. Provi 0015 G. Pvrenacantha (Icacinaceae). sional Patent Application Serial No. 60/161,460 filed (unknown date) and is a continuation patent application 0016 Camptothecin is commonly obtained from the fruit based on U.S. patent application Ser. No. 09/696,042 filed of the Xi Shu tree which grows primarily in China because Oct. 25, 2000. it is widely believed that the fruit of the Xi Shu tree contains the highest levels of camptothecin. The Xi Shu tree (Camp BACKGROUND OF THE INVENTION totheca acuminata) is a species of the genus Camptotheca, which is a Chinese endemic genus of the family Comaceae 0002) 1. Field (or Nyssaceae). The Xi Shu trees are processed and then 0003. The present invention relates to a process for chemical Solvents are used to obtain Small amounts of increasing the amount of indole and quinoline alkaloids, camptothecin from large quantities of the processed bark, particularly camptothecins and related compounds harvested fruit, leaves, and stems. Accordingly, the cost of pharma from plants, more specifically, the present invention is ceutical grade camptothecin is quite high. This high price described in terms of harvesting increased amounts of escalates the cost of camptothecin-based medications. So alkaloids from plants by increasing the growth of young far, there has been no Success in Synthesizing camptothecin tissues having high concentrations of the alkaloids and by at commercially acceptable levels. Additionally, it has been the use of trichome management techniques based on inter found that cell cultures have not been able to produce a action principles of hormones and alkaloids in plants. consistently higher yield of camptothecin than has been obtained from trees of Camptotheca acuminata. 0004 2. Background 0017 Further complicating the problem is the fact that 0005 The plant-produced alkaloid camptothecin (CPT) the Chinese government has declared Camptotheca acumi is presently becoming widely used in anti-cancer medica nata as an endangered species and accordingly has limited tions. Specifically, camptothecin is used in production of the harvest of these plants growing in the wild. Topotecan (TPT, Hycamtin(R) for the treatment of advanced ovarian cancer and Small cell lung cancer, Irinotecan (CPT 0018. Therefore, a need has developed in the art to 11, Camptosar®) for the treatment of colon cancer, and develop a system for increasing the production of camptoth 9-Nitrocamptothecin (9-NC, Rubitecan(R) for the treatment ecin in plants to meet the growing demand for camptothecin of pancreatic cancer. It has been reported that additional for use in cancer-fighting medications. drug Studies are presently underway to expand the use of this important plant alkaloid into Still other anti-cancer drugs. SUMMARY 0019. The present invention provides a system for 0006 A diagram of the chemical structure of natural increasing the production of the plant-produced alkaloid camptothecin and its analogs appears in FIG. 1. camptothecins in those species of plants which produce 0007 Since the initial identification of camptothecin, at camptothecins according to interaction principles of hor least 11 different naturally occurring camptothecin analogs mones and alkaloids. Specifically, the System of the present have been identified from wood, bark, and fruits of Camp invention is directed to: totheca acuminata plants. Specifically, 10-hydroxycamp tothecin is found in Wood and in fruits, 10-methoxycamp 0020 (1) Accelerating the growth of young vegeta tothecin is also found in Wood, 11-hydroxycamptothecin is tive tissues, particularly leaves and Stems, found in fruits, 11-methoxycamptothecin is also found in 0021 (2) Inducing the production of camptothecins fruits, 20-deoxycamptothecin is found in bark, 20-hexanoly bearing glandular trichomes on young vegetative camptothecin is also found in bark and 20-hexanoyl-10 tissues, particularly leaves and Stems, by the appli methoxycamptothecin is found in bark, 22-hydroxyacumi cation of trichome management techniques including natine is found in fruits, 19-hydroxymappicine is found in T-pruning, Simulated herbivory or leaf tip pinching fruits, and 19-O-methylangustoline is also found in fruits. and the imposition of environmental StreSS factors on 0008 Many plant species which are known to produce the plants, camptothecins (the term camptothecins is used to describe camptothecin and its analogs). The plants which are known 0022 (3) Maximizing the amount of camptothecins to produce camptothecins are: bearing glandular trichomes obtained from the plants by harvesting intact clippings of young vegetative 0009 A. Camptotheca (Nyssaceae); including: tissues at predetermined times, Camototheca acuminata Decaisne, Camptotheca acuminata var. tenuifolia Fang et Song, Camptoth 0023 (4) Preserving the camptothecins-bearing tri eca acuminata var. rotundifolia Yang et Duan, chomes on the harvested young vegetative tissues to Camptotheca vunnanensis Dode, Camiptotheca low maximize the amount of camptothecins-bearing reyana Li, Camptotheca lowreyana Li Katie; glandular trichomes remaining with the young Veg etative tissues; 0010) B. Ervatamia (Apocynaceae); 0024 (5) Treating the young vegetative tissues with 0011 C. Nothapodvtes (Mappia) (Olacaceae); either an ultraSonic processor, a homogenizer, or US 2004/0058818A1 Mar. 25, 2004

other mechanical device to break the walls of the both indoleacetic acid (Route A for Stimulating growth) and glandular trichomes to release the camptothecins camptothecin (Route B for inhibiting growth); contained therein. 0036) FIG. 6 is a photograph of Camptotheca lowreyana 0.025 My invention is useful for any plant matters con "Katie; taining alkaloids, including indole and quinoline alkaloids, 0037 FIG. 7 is a drawing of a Camptotheca leaf after particularly camptothecins. These plant matters may be of leaf-tip pinching; pure genetic origin, mixed genetic or hybrid origin, or unknown genetic origin. Plant matters may be harvested 0038 FIG. 8 is a drawing of Camptotheca seedling from plants of either natural Species and varieties or culti T-pruning treatments and control; vated varieties (cultivars) growing in both the natural and 0039 FIG. 9 is a drawing of the leaf-tip pinching tech cultivated conditions. nique as applied in Camptotheca, 0026. Accordingly, it is an object of the present invention to induce the production of camptothecins-bearing glandular 0040 FIG. 10 is a picture of HPLC profiles showing the trichomes and to preserve and extract the camptothecins induction of CPT and its analogs in Camptotheca acuminata from plants. by pinching (a: control, b: with pinching treatment); 0027. It is also an object of the present invention to 0041 FIG. 11 is a scanning electron micrograph of a induce the production of camptothecins by managing the glandular trichome on upper leaf Surface of Camptotheca production of the plant hormones which affect the produc acuminata (A. before treatment, B: after treatment); tion of camptothecins by the plant. 0042 FIG. 12 is a table stating the mean height growth 0028. It is also an object of the present invention to of plants with different T-pruning treatments, accelerate the growth of young vegetative tissues, Specifi 0043 FIG. 13 is a table listing the mean branch number cally leaves and Stems, and to further induce the production of plants with different T-pruning treatments, by the plant of camptothecins-bearing glandular trichomes on the accelerated growth vegetative tissues. It is still a 0044 FIG. 14 is a graph of the monthly biomass pro further object of the present invention to provide a method duction of intact young tissues with and without T-pruning; for the harvesting and the preservation of young vegetative 004.5 FIG. 15 is a table noting the effects of T-pruning tissues So that the loSS of camptothecins-bearing trichomes treatments on CPT contents of intact young tissues of is minimized. Camiptotheca acuminata, 0029. It is yet another object of this invention to provide 0046 FIG. 16a is a graph of the effect of pinching on a method for the effective extraction of camptothecins from camptothecins-bearing glandular trichomes by breaking the CPT concentration in the whole plant of Camptotheca trichome walls. acuminata, 0047 FIG. 16b is a graph of the effect of pinching on BRIEF DESCRIPTION OF THE DRAWING CPT concentration in intact young tissueS of Camptotheca FIGURES acuminata, 0.030. A more complete understanding of the objects and 0048 FIG. 16c is a graph of the effect of pinching on processes of the present invention may be had by reference CPT yield on the whole plant in Camptotheca acuminata, to the following detailed description taken in conjunction with the accompanying drawings, wherein: 0049 FIG. 16d is a graph of the effect of pinching on CPT yield on intact young tissues in Camptotheca acumi 0.031 FIG. 1 is a set of diagrams of the chemical struc nata, tures of natural camptothecin and its analogs in Camptoth eca acuminata camptothecin (1), 10-hydroxycamptothecin 0050 FIG. 17a is a graph of CPT induction by pinching (2), 10-methoxycamptothecin (3), 11-hydroxycamptothecin in young leaves and relatively young leaves of Camptotheca (4), 11-methoxycamptothecin (5), 20-deoxycamptothecin acuminata, (6), 20-hexanoylcamptothecin (7), 20-hexanoyl-10-meth 0051 FIG. 17b is a graph of CPT induction by pinching oxycamptothecin (8), 22-hydroxyacuminatine (9), 19-hy in old leaves of Camptotheca acuminata, droxymappicine (10), 19-O-methylangustoline (11), and Vincoside-lactam (12); 0.052 FIG. 17c is a graph of CPT induction by pinching in young Stems and old Stems in Camptotheca acuminata, 0032 FIG. 2 is a table listing CPT distribution in differ ent tissueS of Camptotheca acuminata, 0053 FIG. 17d is a graph of CPT induction by pinching in young roots and old roots in Camptotheca acuminata, 0.033 FIG. 3 is a scanning electron micrograph of Camp totheca lowreyana Katie': a... the surface view of lower leaf 0054 FIG. 18 is a table of the effects of pinching epidermis, b. a mature glandular trichome (GT) on the lower treatments on CPT contents of intact young tissues of leaf Surface; Camptotheca acuminata under irrigation System; 0034 FIG. 4 is a table stating the glandular trichome size 0055 FIG. 19 is a table of the mean growth of one-year and density on lower leaf surfaces and CPT concentrations old Seedlings grown under different light levels, in leaves of Camptotheca, 0056 FIG. 20 is a table of the mean growth and glan 0.035 FIG. 5 is a diagram of two biosynthetic pathways dular trichome density of three-year-old Seedlings grown showing tryptophan (Trp) as a biosynthetic precursor for under different light levels; US 2004/0058818A1 Mar. 25, 2004

0057 FIG. 21 is a table of the production of biomass and tradictory results in the literature. Specifically, Studies have CPT of intact young tissues under different water conditions; shown that almost all parts of the plants in Camptotheca acuminata would yield the alkaloid camptothecin with con 0.058 FIG. 22 is a table of the distribution pattern of centrations ranging from 0.004% to 0.400% per dry weight biomass, CPT content, and CPT yield in an intact clipping of vegetative biomass. Other studies indicated that the of Camptotheca acuminata, content of the alkaloid camptothecin in different parts of 0059 FIG. 23 is a drawing showing the distribution of Camptotheca acuminata occur at an average rate of biomass and CPT yield in an intact clipping (intact young 5:10:5:2:15 for roots: root bark: stem bark: stems: and fruits, tissue) in Camptotheca acuminata, respectively. Accordingly, many believe that the fruit 0060 FIG. 24a is a graph of the effect of harvest cycle includes the highest level of camptothecin. on CPT content of intact young tissues in Camptotheca 0073. In an effort to better determine the location of the acuminata, camptothecins in Camptotheca acuminata, Some researchers found that the amount of camptothecin contained in the 0061 FIG. 24b is a graph of the effect of harvest cycle young leaves was approximately 50% higher than the on CPT yield of intact young tissues in Camptotheca acumi amount of camptothecin found in the fruit of the plant and nata, as much as 250% higher than the amount of camptothecin 0062 FIG. 25 is a graph of the variation in CPT con found in the bark. It is now widely accepted by those who centration of young leaves with tree age in Camptotheca Study Camptotheca acuminata that the young leaves of the acuminata, plant produce more camptothecin than the older leaves. However, it has never been clearly understood exactly in 0063 FIG. 26a is a graph of the monthly change of CPT which cells and by what pathways camptothecins are accu content of intact young tissueS of Camptotheca acuminata, mulated in the plants. 0064 FIG. 26b is a graph of the monthly yield of CPT of 0074 AS previously indicated, camptothecin is an alka intact young tissueS of Camptotheca acuminata, loid. An alkaloid is a Secondary metabolite which is pro 0065 FIG. 27 is a table of the CPT preservation in intact duced by plants. For many years it was believed that young tissueS preserved by different methods, Secondary metabolites had little explicit function in plants. However, recent research has indicated that Secondary 0.066 FIG. 28a is a graph of the effect of homogenizer metabolites play a significant physiological and ecological treatment on CPT extraction in Camptotheca acuminata by role in plants. It is known that in Some plants, particularly duration of treatment time, and higher plants, the plant produces glandular trichomes which, 0067 FIG. 28b is a graph of the effect of homogenizer in addition to other parts of the plants, contain Secondary treatment on CPT extraction in Camptotheca acuminata by metabolites, Specifically alkaloids, to defend against insect extraction efficacy. herbivory and attacks on the plant by microbes. 0075 My research to determine the accumulation sites of DESCRIPTION OF THE EMBODIMENTS the camptothecins in the leaves of plants in Camptotheca 0068 Abetter understanding of the discoveries which led acuminata began with an analysis of young leaves. This to the creation of the disclosed System for increasing the analysis led to the discovery that the young leaves of production of camptothecins from plants may be obtained Camptotheca acuminata contained a three to five times from an understanding of the research and development higher concentration of camptothecin than existed in older history of the disclosed invention. leaves. This discovery was consistent with the well known finding that Some alkaloid-containing plant Species have 0069. Historical Background higher alkaloid levels in young leaves than in older leaves. 0070 The discovery which led to the use of the alkaloid It was still unknown at this point in the research exactly camptothecin as the basis for anti-cancer drugs may be where the camptothecin was contained in the leaf. traced back to reports that the leaves of the Camptotheca 0076. The economic significance of trichome response acuminata tree had anti-cancer activity. However, early and the chemical defense response of plant mechanical or research on the presence of camptothecin in Camptotheca environmental Stresses have remained unexplored. Plant acuminata produced inconclusive results. Specifically, Some growth management Strategies to investigate the maximiza researchers were unable to detect camptothecin in the leaves, tion of the Stimulation of chemical production in medicinal while others believe that more camptothecin was contained plants have not been developed. Accordingly, to determine in the leaves of the plant than in either the roots or the Stems. how to cause plants in the Camptotheca genus to produce What was known is that the leaves of the Camptotheca more camptothecins, it was necessary to discover those acuminata plants were toxic and that goats who ate the Strategies which Stimulated the production of camptothecin leaves of the plants repeatedly died. in plant trichomes. 0.071) I found still further evidence concerning the loca 0.077 On the Timing of the Presence of Camptothecins in tion of camptothecin in Camptotheca acuminata from the Plant Life fact that the Chinese Tung people in Guangdong province 0078. To further understand the biosynthesis of the camp had been using the extract of young leaves of trees of tothecins in the leaves, it was also necessary to add a time Camptotheca (genus) with alcohol as a treatment for Stub component to the description of a leaf. Specifically, it was born skin diseases, including cancers, for many years. necessary to define young leaves as leaves that were newly 0.072 An initial investigation into the yield of the alka Spread, Specifically, young leaves that were mostly less than loid camptothecin by Camptotheca acuminata revealed con one week old. US 2004/0058818A1 Mar. 25, 2004

0079. By doing further analysis on the tissues of the the glandular trichomes found on the leaves of plants in the young leaves, the Stems attached to young leaves, and the Camptotheca genus were the accumulation site for the mature bark of the trees in Camptotheca acuminata, it was camptothecins. discovered that the tissueS of the young leaves, the Stems 0084. Initially, I discovered that there was a positive attached to the young leaves, and the bark of young Stems of correlation between the concentration of camptothecins and the trees in Camptotheca acuminata produce higher con the glandular trichome density and glandular trichome size centrations of camptothecins than the tissueS of older leaves, on leaf Surfaces for three species of camptothecins bearing the Stems attached to older leaves, and the bark of old stems plants as shown in FIG. 4. It is also known that the young in Camptotheca acuminata as shown in FIG. 2. Specifically, leaves and Stems in Camptotheca plants have the highest it was discovered that the bark of young branches had about photosynthetic rates and are production sites of Some hor a 100% higher concentration of camptothecin than the wood mones Such as auxin and thus contributed primarily to the on the interior part of the tree. In contrast, it was also found growth of the plant. Further, it was known that young tissues that tissues from the wood, the root, and the fruit of older contain more tasteful materials than the older tissues and trees showed a different pattern. Specifically, it was also thus provided food of better quality for herbivores. I there found that the concentration of camptothecin in young roots fore determined that young tissues were the most valuable is only about 32.5% of the concentration of camptothecin in for plant growth and at the same time the most Vulnerable to the roots of trees. It was further found that the concentration herbivory. of camptothecin in young fruit at the flower Stage is only 0085. According to theories used by botanists, the most about 45% of the concentration of camptothecin in mature valuable part of a plant should be that part of the plant which fruits. (As shown in FIG. 2, a mature fruit is 16 weeks old requires the most protection against herbivores. Therefore, I or older.) These patterns of distribution of camptothecin led expected that the young leaves of plants in the Camptotheca to further investigation as to the exact site and the timing of genus should contain higher levels of camptothecin than the the accumulation of camptothecins in Camptotheca acumi older leaves. Field observations revealed that the young FitIt(i. leaves and Stems are not often attacked by herbivores. I also 0080 Trichomes and Camptothecins observed, through microscopic analysis, that the density of the glandular trichomes on young leaves and StemS is much 0.081 Trichomes are known to be hair-like appendages higher than the density of glandular trichomes on old leaves which extend from the epidermis of the aerial tissues of and stems. Further analysis of the plant revealed that glan plants. Trichomes were one of the first anatomical features dular trichomes were not found in the interior parts of the discovered by early researchers on plants. Trichomes may be plant, Specifically the wood. Furthermore, it was found that unicellular or multicellular glandular or non-glandular struc there were low concentrations of camptothecins in the tures and may be of Several morphological types, Specifi interior parts of the plants, although parenchymas in these cally, Straight, hooked or Stellate. interior parts may store certain amounts of camptothecins at certain times of plant development. 0082 Glandular trichomes are quite common in flower ing plants. A variety of natural products, including alkaloids, 0086. Because trichomes are not visible to the naked eye, terpenoids, and phenolics, accumulate in the glandular tri it is difficult to do a direct analysis of the amount of chomes of Some plants, and many of these Substances appear camptothecins in microscopic trichomes. In Searching for an to be produced and stored within the trichomes themselves. alternative method for determining whether or not camp It is also well known that trichomes can Serve to protect tothecins were in the microscopic trichomes, I determined plants from damage by herbivores. However, Studies leading that when the leaf and Stem Surfaces were illuminated using to this conclusion are largely limited to major vegetable or 360 nm ultraViolet light, trichomes, particularly glandular commercial crops or Specifically Species with large glandu trichomes, appeared with intense blue fluorescence. This lar trichomes Such as Nictotiana and Arabidonsis. Other intense blue fluorescence indicated that there were highly research on trichomes has been directed to enhancing bio concentrated alkaloids, including camptothecins, in the tri logical control of the plant or improving the resistance of a chomes, particularly the glandular trichomes. Specifically, plant to pests or diseases. this led to my discovery that camptothecins are mainly accumulated in the large vacuole or cavity within the micro 0083) My microscopic analysis of the leaves of plants of Scopic glandular trichomes on the Surfaces of the leaves and Camptotheca (genus) revealed that the upper leaf and stem Stems, particularly in the Surfaces of young leaves and Surfaces were covered with wax and only sparsely populated Stems, more particularly young leaves and stems that were with global glands. However, it was found that present on less than four weeks old. the lower surfaces of the leaves of the plants in the Camp totheca genus were a plurality of trichomes as shown in 0087 Still further research into the leaves of young plants Scanning electron micrographs at FIG. 3. These trichomes in the Camptotheca genus revealed that the concentration of included Simple unbranched non-glandular hairs and exter camptothecin in leaves showed a different distribution pat nal unicellular glands. AS previously indicated, research tern as the plant leaves developed and matured. Specifically, revealed that in other plants Such as Nicotiana and Solanum, I discovered that the concentration of camptothecin in a leaf glandular trichomes accumulate large quantities of Second decreases as the leaf grows: 1.0:0.5:0.2 (young leaves: ary metabolites. It is believed that these secondary metabo relatively young leaves: old leaves), as opposed to the yield lites are responsible for the plant's defense against herbi of camptothecin per leaf which shows first an increase and Vores. Because it was known that camptothecin was toxic to then a decrease as the leaf grows: 1.0:3.5:2.0 (young leaves: animals and further that camptothecin could be used as an relatively young leaves: old leaves). Accordingly, in young insect chemosterilant, the question remained whether or not leaves (

0107. On Environmental Factors and the Production of hypothesis argues that Secondary metabolites accumulate Plant Alkaloids because of “overflow' metabolism, and emphasizes differ ential enzyme compartmentalization and regulation. Both 0108) Some researchers have hypothesized that a number carbon/nutrient and Substrate/enzyme hypotheses present of environmental factors influence either a plant's trichome induced metabolites as being essentially “waste products', density or its chemical response. neither hypothesis precludes the “defensive' sculpting of the 0109) Water Stress overflow metabolites. 0110. The contribution of trichomes to drought avoidance 0115 Demand-Side Response Hypothesis may be critical under Some circumstances. It has been reported that trichomes improve leaf water Status by entrap 0116. The demand-side hypotheses of how secondary ping and retaining Surface water, thus assisting in the final metabolites change after damage to a plant posits that absorption of water into the mesophyll in Phlomis fruticosa. damage results in biological signals within the plant that On the other hand, water stress in both soil and air may directly regulate Secondary metabolism. The demand-side Stimulate trichome formation in Some plants. For example, hypothesis is built on the premise that concentrations of it has been reported that wheat has significantly denser Secondary metabolites are mostly Strongly influenced by the trichome growth under low Soil moisture conditions. Simi plant's need or demand for defense. The generalized StreSS larly, it has been reported that Severely water-Stressed Taxus response theory argues that plants have hormonally medi X media Hicksii produced significantly more taxanes and ated centralized System of physiological responses for cop ABA than did the less water-stressed plants. Another ing with many diverse Stresses. Optimal defense theories researcher found that Sinapis arvensis might increase tri predict that the most valuable parts of a plant should be most chome density to decrease water loSS under drought. In protected against herbivores. The value of a plant part is contrast, conditions of low evapotranspiration (high humid defined by its contribution to the overall fitness or health of ity and low temperature) not only could slow but also may the plant. During the reproductive phase, flowers and Seeds even truncate the induced response because low evapotrans represent the closest approximation to fitness. Indeed, it has piration will greatly slow the water loSS from plants and been found that flowers and Seeds often contain the highest consequently the transport of alkaloids to the leaves from the levels of defensive secondary metabolites within the plant. roots. It has also been reported that flooding led to signifi During the vegetative phase, young, fully expanded leaves cant decreases in leaf biomass but had no effect on camp have the highest photosynthetic rates and thus contribute tothecin concentration in leaves or Stem in Camptotheca most to growth. It is also found that these young leaves often acuminata. contain more defense chemicals than older leaves. It is also believed that there are two systems which affect the optimal 0111. On the Mechanisms of Plants in Response to Stress distribution of alkaloid defense Systems in tobacco plants, a 0112 Current hypotheses to explain how secondary damage response System that triggers an increase in root metabolites change after damage to a plant can be classified alkaloid Synthesis, and an internal plant plumbing System as either Supply-Side or demand-side. that ensures greater transport of alkaloid-containing xylem fluid to younger leaves. 0113 Supply-Side Response Hypothesis 0117 The active defense response theory is similar to the 0114. The Supply-side hypotheses posits that secondary demand-Side hypothesis but posits far more specificity in the metabolites accumulate in response to imbalances between plant's signaling System. The active defense response theory growth-related processes and metabolite production. postulates that endogenously produced plant damage Signals According to the Supply-Side hypothesis, plants do not or plant damage Signals specific to the invading organism regulate the production of Secondary metabolites to any activate Specific defense responses in a plant. The rapidly extent, and plant defenses are most influenced by the avail induced increases in the production of Secondary metabo ability or Supply of Secondary metabolites within the plant. lites in plants result from Specific Signals which control the The growth/differentiation balance hypothesis posits that all metabolic pathways that produce the chemical defense Secondary metabolites have an ontogenetically determined response in plants. It is found that Sucrose, the major form phenology and that their Synthesis is emphasized during of Sugar transport in the vascular System of a plant (phloem) periods of plant differentiation. The process of plant growth provides a biological Signal. A plant responds to the bio largely occurs during different times than the processes of logical Signal of Sucrose by increasing or decreasing nutrient differentiation that produces resin ducts, trichomes, Spines, flow from the leaves to the roots, the Seeds, and plant Storage and so forth. The “carbon/nutrient” model attempts to organs known as “Sink tissues. RNA molecules may also explain induced changes in Secondary metabolism as a result carry biological Signals long distances within plant via of imbalances between carbon and nutrient requirements for phloem, which has been called the “plant information Super growth and the availability of these resources from the highway.” external environment. According to this hypothesis, only when resources exist in excess of growth requirements are 0118 Because the active defense theory is the most likely they shunted into secondary metabolism. Plants with an model, it can be used to determine how to Stimulate the excess of carbon relative to nutrients are predicted to have production of camptothecins from plants. Accordingly, hor reduced nitrogen-based Secondary metabolites Such as alka mones play more important roles in trichome formation and loids. In contrast, environmental factorS Such as nitrogen alkaloid biosynthesis and transport in plants than previously fertilization and Shade that leaves plants with shortages of known or expected. To understand the correlation between carbon relative to nutrients are predicted to increase nitro hormones and Secondary metabolites, Specifically alkaloids, gen-based Secondary metabolites and reduce carbon-based is critical to developing Strategies for inducing the produc secondary metabolites. The “substrate/enzyme imbalance” tion of alkaloids in plants. It is difficult to present one model US 2004/0058818A1 Mar. 25, 2004

for the correlation between hormones and the formation of herein. This new cultivar is distinguished by its vigorous and trichomes or the production of alkaloids in all plants because dense multi-branching growth habit and its Small and lan different species respond differently and different hormones ceolate or elliptical leaves (shown in FIG. 6) with smooth may have different mechanisms which affect individual margins in both the juvenile and mature Stages of plant alkaloids. In Some cases, alkaloids may also be important in growth. It was found that the Katie cultivar had a signifi hormone biosynthesis and transport. It is my conclusion that cantly higher yield of camptothecins. Specifically, it was the growth promoting hormones (auxins, gebberellins, and found that this new cultivar produced a significantly higher cytokinins) inhibit biosynthesis of alkaloids while other yield of camptothecin (0.1064% on a fresh weight basis) in hormones (ABA and ethylene) stimulate the biosynthesis of its leaves and is more hardy and drought tolerant than any alkaloids. For example, removal or decrease of auxin (IAA) naturally occurring Camptotheca variety. will enhance the yield of indole and quinoline alkaloids in Some plants (e.g. Catharanthus roseus and Camptotheca 0.125 Strategy-Pruning Techniques acuminata). It is not only true in cell culture of Catharan 0.126 The second strategy for stimulating the formation thus roseus cell culture, but it was also found valid in of trichomes and the production of camptothecins involved experiments with Camptotheca plants. the development of T-pruning techniques to decrease auxin 0119) It is my belief that the interrelationship of hor levels within the plant. mones and alkaloids is also present in the production of 0127. On Pruning in General alkaloids by cell culture for all plants. It is critical to balance the amounts of hormones to Successfully induce alkaloid 0128. It is well known that the pruning of a plant is the production in plant cell cultures. AS previously indicated, process of removing a part or parts, living or dead, from a IAA is produced in Shoot apical meristems, developing living plant. The main purpose of pruning is to keep plants leaves, and reproductive buds and it has functions of cell healthy and attractive, to control plant size, to improve the elongation and inhibition of growth of lateral buds (domi quality of flowers, fruits, foliate or Stems, and to reduce nance). Thus, a plant's response to damage is determined by hazards to the plant. age and location of tissues, not merely by how the tissue is 0.129 Pruning is a technique that has long been used in removed or how much of the tissue is removed. horticulture and forestry on landscape plants, fruit plants, 0120 My experiments showed that any treatments which and timber trees worldwide. It is well known that pruning, removed or decreased IAA in plants (e.g., pruning shoot tips if properly done, can greatly increase the biomass growth of particularly the dominant shoots, pinching leaf tips) will a plant over an un-pruned plant. Specifically, it has been Stimulate the production of camptothecins in Camptotheca. reported that removal of the five uppermost immature leaves This explains why there is no inducing the production of or short tipping after 20 to 25 cm. of terminal growth in alkaloids in plants by Some other pruning, pinching, or apple trees can produce more lateral bud break than are herbivory treatments that do not involve the young Stem and produced on non-treated Shoots. leaf tips. This also explains why multiple shoot or slow 0.130 Apical dominance is a basic principle of plant growing plants usually have higher alkaloidal yield. It is not pruning. The terminal bud on a plant produces the hormone clear how hormones inhibit the indole/quinoline alkaloids in auxin which inhibits the growth and development of lateral plants. But tryptophan is a biosynthetical precursor for both buds. When the terminal bud on a plant is removed by indole/quinoline alkaloids and IAA as shown in FIG. 5. The pruning, the lateral buds and Stems below the terminal bud IAA route represents Stimulating growth while the camp grow vigorously. Because auxin moves downward in the tothecin route represents inhibiting growth. Accordingly, I shoot of the plant, apical dominance is strongest in the discovered that camptothecins act in plants just like a vertical or upright shoots or limbs of the plant. For vertical hormone. Some indole and quinoline alkaloids inhibit the shoots or limbs, the most vigorous new growth occurs within cell growth through DNA Topo I enzyme or production of Six to eight inches of the pruning cut. However, regrowth on auxins in plants and thus are cytotoxic (anti-tumor). Camp limbs having a 45 to 60° angle from the vertical will tothecins, vinblastine, and Vincristine are examples. Some develop farther away from the pruning cut. environmental stresses (drought, extreme temperatures, and radiation) may stimulate alkaloidal production due to 0131 On Pruning Techniques increase stress hormone (ABA) level. My experiments 0132) There are several basic types of pruning techniques showed all these Stresses have a positive effect on inducing commonly used in landscape plants. The type of pruning the formation of trichomes and accordingly the production called thinning is the removal of connecting branches at of camptothecins in Camptotheca. their point of origin or shortening the length of a stem by a 0121 On Increasing the Production of Camptothecins lateral cut. Thinning will make a plant grow taller and more open. Heading (also known as topping, rounding over, 0122) To date, there are no studies available on the dehorning, capping and hat-racking) is the pruning of large biological mechanism for induction of camptothecins pro upright Stems between their nodes to reduce the height and duction in Camptotheca. increase the density of the plant. A Selected heading cut removes a large portion of a stem, while a non-Selective 0123 Strategy-Develop a New Cultivar heading cut (also known as Shearing) removes all the stem 0.124. The first strategy to induce the formation of tri tips on a level plane. Tipping is the practice of cutting lateral chomes and the production of camptothecins in plants Stems between nodes to reduce crown width. Both heading involved the creation of a new cultivar. This cultivar, Camp back and tipping are recognized as poor plant maintenance totheca lowreyana Katie, is disclosed in my co-pending techniques which harm trees and should not be used in plant patent application which is incorporated by reference regular tree pruning. US 2004/0058818A1 Mar. 25, 2004

0.133 Also, it has been reported that stem tipping has potential of frost damage. The Systematic pruning and resulted in the highest number of Stems and greatest total frequent harvesting of young tissues from Camptotheca Stem length. In tea bushes, pruning is an essential agronomic minimizes the reduction of the next year's reproductive practice to maintain the tea bushes in a manageable condi growth and thus increases the vegetative growth or biomass tion for plucking. However, it is not clear if pruning induces of leaves and Stems and the accumulation of camptothecins chemical production in medicinal plants. Existing data on in the leaves and Stems of the plant. the effect of pruning on the chemical production in medici 0143. It is well known that the leaves of a plant are nal plants is contradictory. regulators of the plant's response to StreSS. It is well known 0134) Pinching that the production of trichomes, and accordingly, Secondary 0135 The technique called pinching is a common tech metabolites by plant leaves can be induced by herbivory. nique for training perennial herbaceous plants. In pinching, The leaves of Camptotheca plants can be attacked by insect larvae or Small animals. Such as goats or deer. In Some cases, the thumb and forefinger is used to remove very Soft growth; new, almost fully spread leaves and Stems, are more toxic typically, whole buds, leaves or Stems throughout the grow after herbivory. Apparently, the presence of toxic camptoth ing Season. The technique of pinching is used to avoid future ecins in young Camptotheca leaves is induced by herbivory. pruning, to redirect growth, to increase the density of the However, herbivory damage to mature leaves does not plant, or to disbud flower and thin fruits. appear to decrease the level of the plant hormone auxin 0136. Rubbing Significantly. As a result, the production of camptothecins by the plant is limited. The content of camptothecin with 0.137 The practice of rubbing refers to rubbing off unde herbivory is not significantly higher than a plant without Sirable buds, Such as Sprouts on the trunk or Scaffold herbivory: 0.0381%+0.0096 (with herbivory) vs. branches on a fruit tree or any young growth that seems to 0.0514%+0.0065 (without herbivory) in young leaves, be growing in the wrong direction. 0.01711%+0.00383 (with herbivory) vs. 0.01790%+0.00132 0138 Studies on the effect of damage by pruning or (without herbivory) in relatively young leaves, and defoliation on the density of trichomes on plants are incon O.OO984%O.OOO89 (with herbivory) WS. clusive. 0.009335%+0.00086 (without herbivory) in young stems. Therefore, it was determined that uncontrolled herbivory is 0.139. On Pruning and Increasing the Production of not an optimum Strategy to enhance the formation of tri Camptothecins chomes and production of camptothecins. 0140. While the young leaves of the Camptotheca acumi nata have been harvested for the production of camptoth 0144. On Specific Pruning Techniques ecin, there is no Suggestion of any effort being made to 0145 The pruning techniques of the present invention, Stimulate or increase the production of camptothecin by the called T-pruning, control the growth of the Camptotheca plant leaves. It was reported that the yield of camptothecin plants to a low and compact form for Stimulating the was 14.58 mg per plant in a six-week period. It was also development of young tissues, the increased formation of reported that the yield of camptothecin was not constant glandular trichomes, and the production of camptothecins. from plant to plant. Specifically, a camptothecin concentra The present T-pruning technique, as shown in FIG. 8, uses tion range (from 0.045% to 0.349% on a dry weight basis) both Summer and fall pruning after a Spring pruning in the was reported. Same year. This Summer and fall pruning Stimulates addi 0141 Because of the reported production of camptoth tional flushes of stem growth in Camptotheca plants and thus ecin in Camptotheca acuminata, another Strategy for Stimu enhances the formation of glandular trichomes and the lating trichome formation and camptothecins production in production of camptothecins in the vegetative tissues. The Camptotheca centered on developing pruning techniques. young Stems on the plants can be damaged by an early frost, Specifically, these pruning techniques were directed to but harvesting avoids this problem. To reduce frost damage, maintaining the Camptotheca plants in a low and compact the first pruning is preferably done in March after the last form to Stimulate both the biomass yield of young vegetative frost. tissues and to increase the amount of camptothecins in these 0146 The disclosed T-pruning techniques can be applied young tissues. to any age Seedling with either Sexual or asexual origin, preferably 1-3 year old healthy seedlings are best for the 0142. It was discovered that the systematic pruning of application of the T-pruning techniques of the present inven young Stems decreased the level of the plant hormone auxin (LAA) and produced a denser, more compact plant with tion. increased biomass production of young Stems and leaves. 0147 The first pruning in the T-pruning sequence should Specifically, by Systematically pruning the plant, that is, take place immediately after the last frost. If plants have no Stem pruning the plant four times during the first year and dormancy or frost damage in certain regions or conditions pinching off the end portion of the leaf as shown in FIG. 7 (e.g., no low temperatures), or, in other words, plants grow before each harvest, and then continually harvesting the during all Seasons, the first pruning can be done in any time plant eight to ten times after the first year of the pruning of any Season and the fourth pruning can be done in less than treatment, additional flushes of Stem growth appeared Sev one year. The first pruning is accomplished by heading back eral times during the year. Because the new plant shoots a young unbranched shoot to less than about 50 cm. of the caused by pruning in the late Summer or pruning in the fall ground with either pruning Shears or lopping Shears. The could easily be damaged by an early frost, late Summer or pruning cut is made on a slight Slant a quarter inch above a fall pruning is generally not Suggested. It was found, how healthy bud. The bud should be facing the direction pre ever, that frequent harvesting of young tissues avoids the ferred for new growth. This pruning technique will force 2-7 US 2004/0058818A1 Mar. 25, 2004 buds back below the cut into Vigorous, upright growth in 1-2 0154 It has been found that each harvest also serves to weeks. Two or three weeks after the heading, root pruning prune the plant and to induce biomass growth and the can be used to produce a more compact plant. Root pruning formation of trichomes as well as the production of camp is accomplished using a Straight-bladed Spade or other tothecins for the next harvest. This continual pruning and mechanical device. One-third of the root System is pruned harvesting creates a Sustainable System for a long-term away, then after four or five weeks a Second one-third of the harvest of camptothecin. root System is pruned away and then 4-5 weeks later, the O155 The effect of the disclosed T-pruning techniques other third of the root System is pruned away. At the same are detailed in Example 1. Note that the content of camp time as the root pruning, the thumb and forefinger or tothecins in intact young tissue from the Camptotheca plants mechanical leaf tip pinching techniques are used, leaving was significantly increased by the disclosed T-pruning 1-2 old leaves per Stem with the pinched leaves still remain method. In addition, the annual biomass yield of young ing on the branches, as shown in FIG. 9. tissue by the plant was also significantly increased by the 0.148. The second pruning is accomplished 12-20 weeks disclosed T-pruning method. Other natural existing camp after the first pruning. The Second pruning may be accom tothecins, including 10-hydroxycamptothecin will be Sig plished using Scissors or shears and heading back Stems with nificantly increased by the disclosed T-pruning method. a cut angle less than 30 from the main stem to 50 cm. from 0156 After each stem pruning or harvest of young tissue, the ground, heading back those stems between 30 and 70 the thumb and the forefinger are used to pinch off the tip or from the main stem to the third buds from the stem tip while about 20% of the whole blade leaf area, leaving about one only tipping the terminal bud from those Stems with angles or two old leaves per branch while leaving the leaves with more than 70 from the main stem. Stubs are rubbed off the the pinched off tips on the stems. It has been found that this plant. After the pruning, the tips of the one or two remaining technique of pinching off the tips of leaves induces both the leaves per Stem are pinched and the previously pinched formation of glandular trichomes and the production of leaves remain on the stem. The undesirable buds are rubbed camptothecins. Accordingly, the disclosed process of using off below the sixth stem. a leaf tip pinching off technique for the pinching off of leaf 014.9 The third pruning occurs 8 to 12 weeks after the tips to imitate herbivory by insects and Small animals Second pruning. induces the formation of trichomes and the production of camptothecins. Previously, pinching has only been used as a 0150. Using scissor-action shears, those stems with common technique related to training herbaceous landscape angles less than 30 from the vertical are headed back from plants where whole buds, leaves, or Stems are removed from the main stem to 50 cm. from the ground, heading back those a plant to avoid future pruning, to redirect growth, to stems between 30 and 70 from the main stem to the third increase the density of the plant, or to disbud flowers and to buds from the tip of the stem while only tipping the terminal thin out fruits. bud from those stems more than 70 from the main stem. Once again, Stubs are rubbed off. After the T-pruning, the 0157. The Plant's Reaction to Leaf Pinching tips of the one or two remaining old leaves per Stem are 0158. It has been found that there are two peaks of pinched off, and the cut leaves are left on the stem. Once Stimulating the production of camptothecins by pinching off again, the undesirable buds below the sixth stem are rubbed the tips of the leaves over a certain time period for any part off. of young plant tissues as shown in Example 2. The existence 0151. Before any early frost, new growth should be of two peaks Suggests that there are two steps of defensive pruned to avoid frost damage. response by the Camptotheca plants to damage, a first emergency defense, and then a long-term defense. The first 0152 The fourth pruning occurs in the second year emergency defense response occurs two or three days after immediately after the last frost. Using Scissor-action shears, the pinching off of the leaf tips. As a result of the first those branches with angles less than 30 from the main stem emergency defense against plant damage, the plant increases are headed back to within 50 cm. from the ground. Those its content of camptothecins in young tissues, particularly stems between 30 and 70 from the main stem are headed young leaves, largely at the cost of the camptothecins in back to the third buds from the Stem tips, while only tipping older tissues. Thus, the total content of camptothecins in a the terminal buds from those stems more than 70 from the whole plant does not change Significantly during the emer main stem. Once again, Stubs are rubbed off. All dead gency defense. Thus, an emergency response by the plant branches are removed. The undesirable buds below the sixth may produce a false increase of camptothecins yield in a Stems are rubbed off. After the pruning, the tips of the one whole plant. After the emergency response by the plant or two remaining old leaves per branch are pinched off, and wanes, the long-term response by the plant occurs about Six the leaves with tips pinched off are left on the trees after the or eight days after the pinching off of the leaf tips. Therefore, new leaves come out. the optimum time for the leaf tip pinching is at least about 0153. After the fourth pruning, the intact young tissues Six to about eight days before each harvest. can be regularly harvested manually or mechanically. Each 0159. The long-term defense produces a greater increase harvest is equivalent to an application of T-pruning. The tips in the Overall amount of camptothecins in both young and of the leaves should be pinched off 2 to 15 days before each old tissues. Thus, the camptothecins content increases Sig harvest. 10% to 30% of leaf blade areas of 20% to 60% of nificantly in the whole plant. To reach the maximum and all of the leaves on each stem should be pinched off with Stable yield of camptothecins, it is preferred to harvest plant fingers or an equivalent mechanical method. This leaf-tip materials after the long-term response by the plant begins. pinching technique may be applied during any year of plant The Start time of the long-term response by the plant may growth. increase with plantage and plant size. Induced production of US 2004/0058818A1 Mar. 25, 2004

camptothecins in the plant is related to the hormone level in months. It has been found that each plant can produce the plant. Thus, the pinching of the leaf tips is more 600-800 mg of camptothecin annually which is about 7 to 9 important than the amount of cellular damage or the amount times of the production of camptothecin using existing of leaf area lost. The pinching off the tips of young leaves methods. reduces the level of the plant hormone auxin and enhances camptothecins production. It also increases the production 0167 On Preserving the Harvested Materials of camptothecin analogs as in FIG. 10. 0.168. Once harvested, the intact clippings can be pro cessed while fresh within the first two days after harvesting 0160 On Environmental Stress Factors and the Produc or after a longer period, up to Several years, if frozen, as tion of Camptothecins shown in Example 8. Specifically, freezing may take place 0.161 AS previously indicated, it has been reported that in a conventional freezer or by placement of the intact environmental Stresses influence the production of Second clippings in liquid Nitrogen. ary metabolites in Some plants. However, these environmen 0169. In most studies involving Camptotheca plants, tal Stresses can have negative effects on plant growth while oven-dried plant materials have been used for determining having a positive influence on the production of Secondary the presence of camptothecin. Contrary to the existing metabolites. Thus, any plant's response to environmental practice of oven-drying, I have found that fresh or frozen StreSSes varies with the environmental StreSS to the plant plant materials from the Camptotheca plants have the high Species. Because of the nature of the Camptotheca plants, est content of camptothecins as compared to air-dried or environmental factors, Such as water StreSS, as shown in oven-dried plant materials. Still further research revealed Example 3, increases the formation of glandular trichomes that glandular trichomes experience leSS damage or destruc and the production of camptothecin. The results of the tion by freeze-drying than by air drying or oven drying at effects of light intensity are also shown in Example 3. 65 C. in experiments to extract the camptothecin from the 0162. On Harvesting Intact Clippings vegetative tissueS of the plant. Specifically, it was found that 0163 To keep harvested plant materials fresh for long the yield of camptothecin was much higher when freeze periods of time and thus better preserve the trichomes which drying techniques were used than with other plant drying contain the camptothecins, it has been found that young methods. leaves should be harvested with stems. The combination of 0170. On Recovery of the Camptothecins young leaves with their attached StemS is termed an intact 0171 The final step involves recovering the camptoth clipping as shown in Example 4. Intact clippings include any ecins from the trichomes. To accomplish this recovery of the clipping in which a Substantial amount of the original foliage camptothecins from the trichomes, it is necessary to break or leaves remain attached to the Stems. The intact clippings the wall of the glandular trichomes found on the young harvested according to the present invention are mostly 3-20 leaves and stems as described in Example 9 and as shown in days old. It has been found that harvesting intact clippings FIG. 11. The walls of glandular trichomes are generally better preserves trichomes and the camptothecins content in found to be much thicker than those of Surrounding plant leaves and Stems. In addition, more camptothecins exists in epidermis. Manual grinding techniques Such as the use of a intact clippings than in old leaves. mortar or grinder do not effectively break the trichome 0164. The harvesting of the intact clippings can be started walls. The use of an ultrasonic processor to break trichome in late March or early April in the second year after the first walls is more effective, as approximately 80% of the plant pruning as shown in Example 5. It is preferred to have 10-12 glandular trichome walls were broken. harvests annually with about two to four weeks as the 0172 The use of a homogenizer having a peripheral harvest cycle. In Some warmer climates more harvests may Speed of 26 meters per Second for 120 Seconds increases the be possible due to the longer growing period. extraction rate of camptothecin. 0.165. It has been reported by others that in Camptotheca 0173 Still other mechanical techniques such as those acuminata the concentration of camptothecin in leaves using the action of moving Small glass spheres may also be declines at 11% each month from April to October, and the camptothecin concentration in leaves decreases significantly used to break the trichome walls to recover the camptoth with tree age; 16 times lower in 4-year-old trees than in ecins. 2-year-old trees. It has been hypothesized that camptothecin What is claimed is: defense mechanisms are programmed for early ontogenic 1. A process for increasing the production of camptoth Stages in Camptotheca acuminata. ecins by a plant comprising the Step of 0166 My study determined that there is no significant physically, biologically, or ecologically controlling the change in the content of camptothecins with tree age using amount of hormones produced by the plant. the pruning and harvesting System of the present invention 2. The proceSS as defined in claim 1 wherein Said physical, as shown in Example 6. Accordingly, Camptotheca trees biological, or ecological control of the amount of hormones pruned and harvested according to the present invention will produced by the plant includes reducing the amount of auxin yield camptothecins for many years. There will be signifi produced by the plant. cant Seasonal change of the content of camptothecins in the 3. The proceSS as defined in claim 2 wherein the amount young tissueS of the plant during the growing Season as of Said auxin produced by the plant is reduced by removing shown in Example 7. The highest camptothecins yield will those sites of the plant which produce Said auxin. occur in the middle of the growing Season. Both camptoth 4. The process as defined in claim 3 wherein the removal ecins yield and the biomass of intact young tissues can be of Said Sites of the plant which produce Said auxin is improved with proper irrigation, particularly during dry accomplished by first pruning during a first year of plant US 2004/0058818A1 Mar. 25, 2004 growth and then periodically harvesting young vegetative 14. A process for increasing the production of camptoth tissues from the plant during a Second and Subsequent years ecins by a plant comprising the Step of of plant growth. increasing the formation of camptothecins-bearing tri 5. The process as defined in claim 4 wherein Said pruning chomes on young vegetative tissueS of the plant by during Said first year of plant growth further includes Stem physically, biologically, or ecologically controlling the pruning four times during Said first year of plant growth. amount of hormones produced by the plant. 6. The process as defined in claim 5 wherein said stem 15. The process as defined in claim 14 wherein said pruning includes: physical, biological, or ecological control of the amount of a first pruning after the last frost, hormones produced by the plant includes reducing the amount of auxin produced by the plant. a Second pruning about 12 to 20 weeks after Said first 16. The process as defined in claim 15 wherein the amount pruning, of Said auxin produced by the plant is reduced by removing a third pruning about 8 to 12 weeks after Said Second those sites of the plant which produce Said auxin. pruning, 17. The process as defined in claim 16 wherein the removal of Said Sites of the plant which produce Said auxin a fourth pruning after the last frost at the end of Said first is accomplished by first pruning during a first year of plant year of plant growth. growth and then periodically harvesting young vegetative 7. The process as defined in claim 6 further including root tissues from the plant during a Second and Subsequent years pruning during Said Second year of plant growth, Said root of plant growth. pruning further including: 18. The process as defined in claim 17 wherein said a first pruning of about /3 of the roots, pruning during Said first year of plant growth further includes Stem pruning four times during Said first year of a Second pruning of about/3 of the roots about five weeks plant growth. after Said first pruning of about /3 of the roots, 19. The process as defined in claim 18 wherein said stem a third pruning of about /3 of the roots about five weeks pruning includes: after Said Second pruning of about /3 of the roots. 8. The process as defined in claim 6 wherein: a first pruning after the last frost, a Second pruning about 12 to 20 weeks after Said first Said first pruning includes heading back Stems to less than pruning, about 50 cm. above the ground; Said Second pruning includes heading back Stems with a a third pruning about 8 to 12 weeks after said Second crotch angle less than about 30 from the main stem of pruning, the plant to about 50 cm. above the ground, heading a fourth pruning after the last frost at the end of Said first back those stems between about 30 and about 70 year of plant growth. from the main stem to the third bud from the stem tip; 20. The process as defined in claim 19 further including Said third pruning includes heading back Stems with root pruning during Said Second year of plant growth, Said angles less than about 30 from the main stem to about root pruning further including: 50 cm. above the ground, heading back the Stems a first pruning of about /3 of the roots, between about 30 and about 70 from the main stem to the third bud from the stem tip; a Second pruning of about/3 of the roots about five weeks after Said first pruning of about/3 of the roots, Said fourth pruning includes heading back Stems with angles between about 30 and about 70 from the main a third pruning of about /3 of the roots about five weeks stem to the third bud from the stem tip. after Said Second pruning of about /3 of the roots. 9. The process as defined in claim 7 further including the 21. The process as defined in claim 19 wherein: step of pinching off about 10% to about 30% of the leaf area Said first pruning includes heading back Stems to less than at the tip of the leaf of about 20% to about 60% of all the leaves on each Stem at the same time as each of Said root about 50 cm. above the ground; prunings during Said first or Subsequent years of plant Said Second pruning includes heading back Stems with a growth. crotch angle less than 30 from the main stem of the 10. The process as defined in claim 4 wherein said plant to about 50 cm. above the ground, heading back periodic harvesting of young vegetative tissues is accom those stems between about 30 and about 70 from the plished at about two to four week intervals during Said main stem to the third bud from the stem tip; Second and Subsequent years of plant growth. 11. The process as defined in claim 10 wherein said Said third pruning includes heading back Stems with periodic harvesting of young vegetative tissues is accom angles less than 30 from the main stem to about 50 cm. plished about 10 to 12 times per year of plant growth. above the ground, heading back the Stems between 12. The proceSS as defined in claim 4 wherein Said young about 30 and 70° from the main stem to the third bud vegetative tissues are between about 3 to about 20 days old. from the Stem tip; 13. The process as defined in claim 12 wherein about 10% Said fourth pruning includes heading back Stems with to about 30% of the leaf area at the tip of the leaf of about angles between about 30 and about 70 from the main 20% to about 60% of all the leaves on each stem is pinched stem to the third bud from the stem tip. off at least about 6 to about 8 days before each harvest of 22. The process as defined in claim 20 further including young vegetative tissues. the step of pinching off about 10% to about 30% of the leaf US 2004/0058818A1 Mar. 25, 2004

area at the tip of the leaf of about 20% to about 60% of all a third pruning of about /3 of the roots about five weeks the leaves on each Stem at the same time as each of Said root after Said Second pruning of about /3 of the roots. prunings during Said first or Subsequent years of plant 34. The process as defined in claim 32 wherein: growth. 23. The process as defined in claim 17 wherein said Said first pruning includes heading back Stems to less than periodic harvesting of young vegetative tissues is accom about 50 cm. above the ground; plished at about two to four week intervals during Said Said Second pruning includes heading back Stems with a Second and Subsequent years of plant growth. crotch angle less than 30 from the main stem of the 24. The process as defined in claim 23 wherein said plant to about 50 cm. above the ground, heading back periodic harvesting of young vegetative tissues is accom those stems between about 30 and about 70 from the plished about 10 to 12 times per year of plant growth. main stem to the third bud from the stem tip; 25. The process as defined in claim 17 wherein said young vegetative tissues are between about 3 to about 20 days old. Said third pruning includes heading back Stems with 26. The process as defined in claim 25 wherein about 10% angles less than about 30 from the main stem to about to about 30% of the leaf area at the tip of the leaf of about 50 cm. above the ground, heading back the Stems 20% to about 60% of all the leaves on each stem is pinched between about 30 and about 70 from the main stem off at least about 6 to about 8 days before each harvest of to the third bud from the stem tip; Said young vegetative tissues. Said fourth pruning includes heading back Stems with 27. A process for increasing the production of camptoth angles between about 30 and about 70 from the main ecins by a plant comprising the Steps of: stem to the third bud from the stem tip. increasing the amount of young vegetative tissueS pro 35. The process as defined in claim 33 further including duced by the plant; the step of pinching off about 10% to about 30% of the leaf increasing the formation of camptothecins-bearing tri area at the tip of the leaf of about 20% to about 60% of all chomes on Said increased amount of Said young Veg the leaves on each Stem at the same time as each of Said root etative tissues by physically, biologically, or ecologi prunings during Said first or Subsequent years of plant cally controlling the amount of hormones produced by growth. the plant. 36. The process as defined in claim 30 wherein said 28. The process as defined in claim 27 wherein said periodic harvesting of Said young vegetative tissues is physical, biological, or ecological control of the amount of accomplished at about two to four week intervals during Said hormones produced by the plant includes reducing the Second and Subsequent years of plant growth. amount of auxin produced by the plant. 37. The process as defined in claim 36 wherein said 29. The process as defined in claim 28 wherein the amount periodic harvesting of Said young vegetative tissues is of Said auxin produced by the plant is reduced by removing accomplished about 10 to 12 times per year of plant growth. those sites of the plant which produce Said auxin. 38. The process as defined in claim 30 wherein said young 30. The process as defined in claim 29 wherein both the vegetative tissues are between about 3 to about 20 days old. removal of those Sites of the plant which produce auxin and 39. The process as defined in claim 38 wherein about 10% increasing the amount of young vegetative tissueS produced to about 30% of the leaf area at the tip of the leaf of about by the plant is accomplished by first pruning during a first 20% to about 60% of all the leaves on each stem is pinched year of plant growth and then periodically harvesting young off at least about 6 to about 8 days before each harvest of vegetative tissues from the plant during a Second and Said young vegetative tissues. Subsequent years of plant growth. 40. A proceSS for increasing the amount of camptothecins 31. The process as defined in claim 30 wherein said bearing trichomes harvested from a plant comprising the pruning during Said first year of plant growth further Steps of includes Stem pruning four times during Said first year of plant growth. increasing the amount of young vegetative tissueS pro 32. The process as defined in claim 31 wherein said stem duced by the plant; pruning includes: increasing the formation of camptothecins-bearing tri a first pruning after the last frost, chomes on Said increased amount of young vegetative tissues, a Second pruning about 12 to 20 weeks after Said first pruning, reducing the amount of camptothecins-bearing trichomes falling away from Said young vegetative tissues after a third pruning about 8 to 12 weeks after Said Second pruning, the harvesting of the young vegetative tissues. 41. The process as defined in claim 40 wherein said a fourth pruning after the last frost at the end of Said first amount of camptothecins-bearing trichomes falling away year of plant growth. from Said young vegetative tissues is reduced by processing 33. The process as defined in claim 32 further including Said young vegetative tissues within about two days after root pruning during Said Second year of plant growth, Said harvesting. root pruning further including: 42. The process as defined in claim 40 wherein the amount of Said camptothecins-bearing trichomes falling away from a first pruning of about /3 of the roots, Said young vegetative tissues is reduced by extracting a Second pruning of about/3 of the roots about five weeks freshly harvested matterS Said young vegetative tissues after Said first pruning of about /3 of the roots, Shortly after harvesting. US 2004/0058818A1 Mar. 25, 2004

43. A proceSS for increasing the amount of camptothecins breaking Said trichome walls to release the camptothecins harvested from a plant which includes camptothecins-bear with the camptothecins-bearing trichomes. ing trichomes comprising the Steps of: 44. The process as defined in claim 43 wherein said trichome walls are broken using ultrasound. increasing the amount of young vegetative tissueS pro 45. The process as defined in claim 43 wherein said duced by the plant; trichome walls are broken using a homogenizer. increasing the formation of camptothecins-bearing tri 46. The process as defined in claim 43 wherein said chomes on Said increased amount of young vegetative trichome walls are broken by a physical impact on Said tissues, trichome walls. reducing the number of camptothecins-bearing trichomes 47. The process as defined in claim 43 wherein said falling away from the young vegetative tissues after the camptothecins are collected in a Solvent. harvesting of the young vegetative tissues; k k k k k